Skip to main content

Full text of "Clinical diagnosis; a manual of laboratory methods"

See other formats 


^^.^^•^^^^K^M.~S^^^S:S^i^^^J^^'^^^ 


Presented  by 
Estate  of  Harry  W,  Forbes,  D.O. 


E     COLLEGE    OF    OSTEOPATHIC     PHYSICIANS     0 
AND  SURGEONS  •    LOS  ANGELES,  CALIFORNIA 


«a$^«a(»«9«3(a(3(d9vd«(3(»9(d($9(S(»S«9aa(»$(»(»99« 


UNIVERSITY  OF  CAMPORNIA 

CALIFORNIA  COLLEGE  OF  MEDICINE 

LIBRARY 

JlIN      1  1^71 

IRVINE,  CALIFORNIA  92664 


Digitized  by  tlie  Internet  Arcliive 

in  2007  witli  funding  from 

IVIicrosoft  Corporation 


littp://www.arcli  ive.org/details/clinicaldiagnosiOOtoddiala 


PLATE  I 


I. 

PALE  YELLOW. 

II. 
LIGHT  YELLOW. 

III. 
YELLOW. 

IV. 

REDDISH   YELLOW. 

V. 

YELLOWISH  RED. 

^^^^^^^^^^^ 

VI. 

RED. 

vir. 
BROWNISH  RED. 

VIII. 

REDDISH  BROWN. 

1 

^^^^^^^^^^^^^^H 

^^^^^^^^^^^^^^^^^^^^H 

Scale  of  urinary  colors,  according  to  \'of;i-l. 


Clinical  Diagnosis 

A  MANUAL  OF  LABORATORY  METHODS 


BY 

JAMES    CAMPBELL    TODD.    Ph.  B..   M.   D. 

PROFESSOR  OF  PATHOLOGY,  UNIVERSITY  OF  COLORADO 


Illustrated 


Second  Edition,  Revised  and  Enlarged 


PHILADELPHIA   AND  LONDON 

W.     B.     SAUNDERS     COMPANY 

1912 


Copyright,  1908,  by  W.  B.  Saunders  Company.     Revised,  reprinted,  and 
recopyrighted  January,  1912 


Copyright,  1912,  by  W.  B.  Saunders  Company 


PRINTED    IN    AMERICA 

PRESS    OF 

W.     B.     SAUNDERS     COMPANV 

PHILADELPHIA 


TO 

MY   FATHER 

THESE   PAGES  ARE 
AFFECTIONATELY    DEDICATED 


'> 


•<\ 


PREFACE  TO  THE  SECOND  EDITION 


While  the  original  purpose  of  this  book — to  present 

^      clearly  and  concisely  the  various  laboratory  methods 

»^AJ      which  are  of  use  in  clinical  medicine — has  not  been  lost 

^      sight  of,  its  scope  has  been  somewhat  enlarged  in  the 

oX      present  edition. 

^  Each  section  has  been  carefully  revised  and  much  new 

'^^       material  has  been  added  to  every  chapter.     Among  the 

'0      many  additions  may  be  mentioned:  the  use  of  artificial 

>v     light  and  the  importance  of  numerical  aperture  in  micro- 

'S^    ^copic  work;  photomicrography  with  simple  apparatus; 

S*        the  antif ormin  method  for  tubercle  bacilli ;  detection  and 

X       significance  of  albumin  in  the  sputum;  Tsuchiya's  modi- 

I        fication  of  Esbach's  test;  the  formalin  test  for  ammonia 

and  Benedict's  methods  for  sugar  in  urine;  volume  index 

Ja      of  red  blood-corpuscles;  Wright  and  Kinnicutt's  method 

i^      of  counting  blood-platelets;  Harlow's  blood-stain;  a  sim- 

"'^     pie  technic  for  the  diagnosis  of  typhoid  fever  by  blood- 

_J     cultures;  the  Wassermann  reaction,  and  Frothingham's 

iaX  impression  method  in  the  diagnosis  of  rabies. 

Because  of  the  growing  importance  of  animal  parasites, 
this  chapter  has  been  entirely  rewritten  and  more  than 
doubled  in  extent.  Two  new  chapters  have  been  added: 
.one  upon  Bacteriologic  Methods,  which  supplements  the 
methods  given  in  other  portions  of  the  book,  and  one 
upon  Preparation  and  Use  of  Vaccines,  including  thera- 
peutic and  diagnostic  use  of  tubercuHn. 

22865 


lO  PREFACE 

Some  of  the  illustrations  have  been  replaced  with  better 
ones  and  many  new  pictures  have  been  added,  including 
eight  photomicrographs  by  Dr.  W.  P.  Harlow,  and  a  con- 
siderable number  by  the  author.  Through  courtesy  of 
Dr.  Langdon  Frothingham,  of  Harvard  University,  a 
colored  plate  showing  Negri  bodies  as  seen  in  his  im- 
pression method  has  been  included. 

The  author  wishes  to  express  his  indebtedness  to  Fran- 
cis Ramaley,  Ph.D.,  Professor  of  Biology  in  the  Univer- 
sity of  Colorado,  and  T.  D.  A.  Cockerell,  Professor  of 
Systematic  Zoology,  for  suggestions  as  to  the  nomen- 
clature of  animal  parasites;  and  to  Dr.  A.  R.  Peebles, 
Professor  of  Medicine,  for  suggestions  and  aid  through- 
out the  revision. 

J.  C.  T. 
Boulder,  Colorado. 


PREFACE 


This  book  aims  to  present  a  clear  and  concise  state- 
ment of  the  more  important  laboratory  methods  which 
have  clinical  value,  and  a  brief  guide  to  interpretation 
of  results.  It  is  designed  for  the  student  and  practi- 
tioner, not  for  the  trained  laboratory  worker.  It  had 
its  origin  some  years  ago  in  a  short  set  of  notes  which 
the  author  dictated  to  his  classes,  and  has  gradually 
grown  by  the  addition  each  year  of  such  matter  as  the 
year's  teaching  suggested.  The  eagerness  and  care  with 
which  the  students  and  some  practitioners  took  these 
notes  and  used  them  convinced  the  writer  of  the  need 
of  a  volume  of  this  scope. 

The  methods  oflfered  are  practical;  and  as  far  as 
possible  are  those  which  require  the  least  complicated 
apparatus  and  the  least  expenditure  of  time.  Simplicity 
has  been  considered  to  be  more  essential  than  absolute 
accuracy.  Although  in  many  places  the  reader  is  given 
the  choice  of  several  methods  to  the  same  end,  the 
author  believes  it  better  to  learn  one  method  well  than 
to  learn  several  only  partially. 

More  can  be  learned  from  a  good  picture  than  from 
any  description,  hence  especial  attention  has  been  given 
to  the  illustrations,  and  it  is  hoped  that  they  will  serve 
truly  to  illustrate.     Practically  all  the  microscopic  struc- 


12  PREFACE 

tures   mentioned,  all  apparatus  not  in  general  use,  and 
many   of  the  color  reactions  are  shown  in  the  pictures. 

Although  no  credit  is  given  in  the  text,  the  recent 
medical  periodicals  and  the  various  standard  works  have 
been  freely  consulted.  Among  authors  whose  writings 
have  been  especially  helpful  may  be  mentioned  v.  Jaksch, 
Boston,  Simon,  Wood,  Emerson,  Purdy,  Ogden,  Ewald, 
Ehrlich  and  Lazarus,  Da  Costa,  Cabot,  Osier,  Stengel, 
and  McFarland. 

The  author  wishes  hereby  to  express  his  indebtedness 
to  Dr.  J.  A.  \\'ilder.  Professor  of  Pathology  in  the  Den- 
ver and  Gross  College  of  Medicine,  for  aid  in  the  final 
revision  of  the  manuscript;  and  to  W.  D.  Engel,  Ph.D., 
Professor  of  Chemistry,  for  suggestions  in  regard  to  de- 
tection of  drugs  in  the  urine.  He  desires  to  acknowl- 
edge the  care  with  which  Mr.  Ira  D.  Cassidy  has  made 
the  original  drawings,  and  also  the  uniform  courtesy  of 
W.  B.  Saunders  Company  during  the  preparation  of 
the   book. 

J.  C.  T. 

Denver,  Colorado. 


CONTENTS 


INTRODUCTION  page 

Use  of  the  Microscope 17 

CHAPTER  I 

The  Sputum 36 

Physical  Examination 38 

Microscopic  Examination 40 

Unstained  Sputum 40 

Stained  Sputum 48 

Chemic  Examination 63 

Sputum  in  Disease 64 

CHAPTER  II 

The  Urine 68 

Physical  Examination 70 

Chemic  Examination 80 

Normal  Constituents 80 

Abnormal  Constituents 99 

Microscopic  Examination 138 

Unorganized  Sediments 141 

Organized  Sediments 151 

Extraneous  Structures 171 

The  Urine  in  Disease 173 

CHAPTER  III 

The  Blood 180 

Hemoglobin 184 

Enumeration  of  Erythrocytes 192 

Color  Index 200 

Volume  Index 200 

Enumeration  of  Leukocytes 202 

Decrease  in  Number  of  Leukocytes 202 

Increase  in  Number  of  Leukocytes 203 

Leukocytosis 203 

Leukemia 208 

Method  of  Counting  Leukocytes 209 

13 


14  CONTENTS 

PAGE 

Enumeration  of  Blood-plaques 213 

Study  of  Stained  Blood 216 

Making  and  Staining  Blood-films 216 

Study  of  Stained  Films 225 

Blood  Parasites 244 

Bacteria 244 

Animal  Parasites 247 

Serum  Reactions 257 

Tests  for  Recognition  of  Blood 274 

Special  Blood  Pathology 275 

Anemia 275 

Leukemia 280 

CHAPTER  IV 

The  Stomach 284 

Examination  of  the  Gastric  Contents 284 

Obtaining  the  Contents 285 

Physical  Examination 288 

Chemic  Examination 289 

Microscopic  Examination 301 

The  Gastric  Contents  in  Disease 304 

Additional  Examinations  which   Give  Information   as  to  the 

Condition  of  the  Stomach 306 

CHAPTER  V 

The  Feces 310 

Macroscopic  Examination 311 

Chemic  Examination 314 

Microscopic  Examination 315 

Functional  Tests 320 

CHAPTER   VI 
Animal  Parasites 323 

Protozoa 326 

Sarcodina 328 

Mastigophora  (Flagellata) 330 

Sp)orozoa 338 

Infusoria 339 

Vermidea , 340 

Platyhelminthes 341 

Nemathelminthes 353 

Arthropoda 366 


CONTENTS  15 

CHAPTER  VII 

Miscellaneous  Examinations 367 

Pus 367 

Peritoneal,  Pleural,  and  Pericardial  Fluids 371 

Cerebrospinal  Fluid 374 

Animal  Inoculation 375 

The  Mouth 377 

The  Eye 381 

The  Ear 383 

Parasitic  Diseases  of  the  Skin 384 

Milk 384 

Syphilitic  Material 388 

Semen , 391 

Diagnosis  of  Rabies 393 

CHAPTER  VIII 

Bacteriologic  Methods 396 

Apparatus 396 

Sterilization • 399 

Preparation  of  Culture-tubes 400 

Culture-media 401 

Staining  Methods 407 

Methods  of  Studying  Bacteria 412 

Characteristics  of  Special  Bacteria 415 

CHAPTER   IX 

Preparation  and  Use  of  Vaccines 419 

Preparation  of  Vaccine 419 

Method  of  Use 425 

Dosage 425 

Therapeutic  Indications 426 

Tuberculins 428 

Tuberculin  in  Diagnosis 429 

APPENDIX 

Apparatus,  Reagents  and  Stains 432 

Apparatus 432 

Reagents  and  Stains 434 

Weights,  Measures,  etc.,  with  Equivalents 439 

Temperature 440 

Index 441 


CLINICAL  DIAGNOSIS 


INTRODUCTION 
USE  OF  THE  MICROSCOPE 

There  is  probably  no 
laboratory  instrument 
whose  usefulness  de- 
pends so  much  upon 
proper  manipulation  as 
the  microscope,  and 
none  is  so  frequently 
misused  by  beginners. 
Some  suggestions  as  to 
its  proper  use  are,  there- 
fore, given  at  this  place. 
It  is  presumed  that  the 
reader  is  already  famil- 
iar with  its  general  con- 
struction (Fig.  i). 

For  those  who  wish 
to  understand  the  prin- 
ciples of  the  microscope 
and  its  manipulation — 
and  best  results  are  im- 
possible without  such  an 
understanding — a  care- 
ful study  of  some  stan- 

2 


Fig.  I. — Handle-arm  microscope:  E,  Eye- 
piece; D,  draw-tube;  T,  body-tube;  RN, 
revolving  nose-piece;  O,  objective;  PH,  pinion 
head;  MH,  micrometer  head;  HA,  handle-arm; 
SS,  substage;  S,  stage;  M.  mirror;  B,  base;  R, 
rack;  P,  pillar;  I,  inclination  joint. 
17 


l8  INTRODUCTION 

dard  work  upon  microscopy,  such  as  those  of  Carpenter, 
Spitta,  and  Sir  A.  E.  Wright,  is  earnestly  recommended. 
It  is  also  recommended  that  the  beginner  provide  him- 
self with  some  slides  of  diatoms,  for  example,  Pleuro- 
sigma  angulalum,  Surirella  gemma,  and  Amphipleura 
pellucida,  costing  fifty  cents  each.  Faithful  practice 
upon  such  test-objects,  in  the  light  of  the  principles  of 
microscopy,  will  enable  the  student  to  reach,  intelli- 
gently, an  accuracy  in  manipulation  to  which  the  ordi- 
nary laboratory  worker  attains  only  slowly  and  by  rule 
of  thumb.  He  will  soon  find  that  the  bringing  of  an 
object  into  accurate  focus  is  by  no  means  all  of  micros- 
copy. 

Illumination. — Good  work  cannot  be  done  without 
proper  illumination.  It  is  difficult  to  lay  too  much 
stress  upon  this  point. 

The  light  which  is  generally  recommended  as  best  is 
that  from  a  white  cloud,  the  microscope  being  placed 
by  preference  at  a  north  window,  to  avoid  direct  sun- 
light. Such  light  is  satisfactory  for  all  ordinary  work. 
Artificial  light  is,  however,  imperative  for  those  who  must 
work  at  night,  and  is  a  great  convenience  at  all  times. 
Properly  regulated  artificial  light,  moreover,  offers 
decided  advantages  over  daylight  for  critical  work. 
Almost  any  strong  light  which  is  diffused  through  a 
frosted  globe  will  give  fair  results.  The  inverted  Wels- 
bach  light  with  such  a  globe  is  excellent.  The  follow- 
ing plan  is  much  used  abroad,  and  gives  results  equal 
to  the  best  daylight:  A  Welsbach  lamp  or  strong  elec- 
tric light  is  used,  and  a  glass  globe — a  six-inch  round- 
bottom  flask  answers  admirably — is  placed  between  it 
and  the  microscope,  to  act  as  a  condenser  (Fig.   2). 


USE  OF   THE   MICROSCOPE 


19 


The  flask  should  be  at  a  distance  equal  to  its  diameter 
from  both  the  Hght  and  the  mirror  of  the  microscope. 
In  order  to  filter  out  the  yellow  rays  the  flask  is  filled 
with  water  to  which  have  been  added  a  few  crystals 
of  copper  sulphate  and  a  little  ammonia. 

For  critical  work,  the  method  suggested  by  Sir  A.  E. 
Wright  is  to  be  preferred.  He  has  shown  that  fog  is 
dispelled  and  definition  is  Improved  if  the  size  of  the 
Hght  source  is  so  regulated  that  its  image,  thrown  upon 


Fig.  2. — Illumination  with  water-bottle  condenser. 


the  slide  by  the  condenser,  coincides  with  the  real  field 
of  the  objective.  Upon  this  principle  a  very  neat  and 
satisfactory  microscope  lamp,  shown  in  Fig.  3,  has  been 
designed  by  B.  H.  Matthews.  It  is  fitted  with  iris- 
diaphragm,  condensing  lens,  small  electric  light,  and 
reflector,  and  has  a  slot  in  which  a  ray  filter  or  ground- 
glass  disc  may  be  inserted. 

Illimiination  may  be  either  central  or  oblique.     Central 
illumination  is  to  be  used  for  all  routine  work.     To  ob- 


20 


INTRODUCTION 


tain  this,  the  mirror  should  be  so  adjusted  that  the  light 
from  the  source  selected  is  reflected  directly  up  the  tube 
of  the  microscope.  This  is  easily  done  by  removing  the 
eye-piece  and  looking  down  the  tube  while  adjusting  the 
mirror.  The  eye-piece  is  then  replaced,  and  the  light 
reduced  as  much  as  desired  by  means  of  the  diaphragm. 
With  daylight,  it  is  best  to  use  the  plane  mirror;  with 
artificial  light,  the  concave  mirror. 


Fig-  3- — Matthews'  microscope  lamp  with  iris-diaphragm. 

Obhque  illumination  is  to  be  used  only  to  bring  out 
certain  structures  more  clearly  after  viewing  them  by 
central  light:  as,  for  example,  to  show  the  edges  of  a 
hyahne  cast  by  throwing  one  of  its  sides  into  shadow. 
Oblique  illumination  is  obtained  in  the  more  simple 
instruments  by  swinging  the  mirror  to  one  side,  so  that 
the  light  enters  the  microscope  obliquely.  The  more 
complicated  instruments  obtain  it  by  means  of  a  rack 


USE   OF   THE   MICROSCOPE  21 

and  pinion,  which  moves  the  diaphragm  laterally. 
Beginners  frequently  use  oblique  illumination  without 
recognizing  it,  and  are  thereby  much  confused.  If  the 
light  be  oblique,  an  object  in  the  center  of  the  field  will 
appear  to  move  from  side  to  side  when  the  fine  adjust- 
ment is  turned  back  and  forth. 

The  amount  of  light  is  even  more  important  than  its 
direction.  It  is  regulated  by  the  diaphragm.  It  is 
always  best  to  use  the  least  light  that  will  show  the  object 


a  b 

Fig.  4. — a,  Hyaline  casts,  one  containing  renal  cells;  properly  subdued  illumination; 
b,  same  as  a;  strong  illumination.  The  casts  are  lost  in  the  glare,  and  only  the  renal 
cells  are  seen.     (From  Greene's  "Medical  Diagnosis"). 

well.  Unstained  objects  require  very  subdued  light. 
Beginners  constantly  use  it  too  strong.  Strong  light  will 
often  render  semitransparent  structures,  as  hyaline  casts, 
entirely  invisible  (Fig.  4).  Stained  objects,  especially 
bacteria,  require  much  greater  light. 

Dark  Ground  Illumination. — This  consists  in  cutting 
out  the  central  rays  of  light  and  directing  the  peripheral 
rays  against  the  object  from  the  side.  Only  those  rays 
which  strike  the  object  and  are  reflected  pass  into  the 
objective.    The  object  is  bright  upon  a  black  back- 


22  INTRODUCTION 

ground.  By  means  of  this  form  of  illumination  very 
minute  structures  can  be  seen,  just  as  particles  of  dust 
in  the  atmosphere  become  visible  when  a  ray  of  sunlight 
enters  a  darkened  room. 

Dark  ground  illumination  for  low-power  work  can  be 
obtained  by  means  of  the  ring  stops  with  central  discs 
which  accompany  most  microscopes  when  purchased. 
The  stop  is  placed  in  a  special  ring  beneath  the  con- 
denser. By  varying  the  size  of  the  central  disc  good 
results  can  be  had  with  the  lower  power  dry  lenses. 

For  oil-immersion  work  a  special  condenser  is  neces- 
sary. With  some  makes  it  is  placed  upon  the  stage  of 
the  microscope;  with  others  it  is  substituted  for  the 
regular  condenser.  It  requires  an  intense  light,  like 
direct  sunlight  or  the  Liliput  arc-light. 

The  chief  use  of  dark  ground  illumination  in  cHnical 
work  is  for  demonstration  of  Treponema  pallidum  in 
fresh  material  (Fig.  158). 

The  Condenser. — For  the  work  of  the  clinical  labora- 
tory a  substage  condenser  is  a  necessity.  Its  purpose 
is  to  condense  the  light  upon  the  object  to  be  examined. 
For  critical  work  the  light  must  be  focused  on  the  object 
by  raising  or  lowering  the  condenser  by  means  of  the 
screw  provided  for  the  purpose.  The  image  of  the  light 
source  will  then  appear  in  the  plane  of  the  object.  This 
is  best  seen  by  using  a  low-power  objective  and  ocular. 
Should  the  image  of  the  window-frame  or  other  nearby 
object  appear  in  the  field  and  prove  annoying,  the  con- 
denser may  be  raised  or  lowered  a  little.  It  is  often 
advised  to  remove  the  condenser  for  certain  kinds  of 
work,  but  this  is  not  necessary  and  is  seldom  desirable. 

It  is  very  important  that  the  condenser  be  accurately 


USE  OF  THE  MICROSCOPE  23 

centered,  and  most  high-grade  instruments  have  center- 
ing screws  by  which  it  can  be  adjusted  at  any  time.  The 
simplest  way  to  recognize  whether  the  condenser  is 
centered  is  to  close  the  diaphragm  beneath  it  to  as  small 
an  opening  as  possible,  then  remove  the  eye-piece  and 
look  down  the  tube.  If  the  diaphragm  opening  does 
not  appear  in  the  center  of  the  field,  the  condenser  is  out 
of  center. 

The  use  of  the  condenser  is  further  discussed  in  the 
following  section. 

Objectives  and  Eye-pieces. — Unfortunately,  different 
makers  use  different  systems  of  designating  their  lenses. 
The  best  system,  and  the  one  chiefly  used  in  this  country, 
is  to  designate  objectives  by  their  focal  lengths  in  milli- 
meters, and  eye-pieces  by  their  magnifying  power, 
indicated  by  an  "  X ."  Most  foreign  makers  use  this 
system  for  their  high-grade  lenses,  but  still  cling  to 
arbitrary  letters  or  numbers  for  their  ordinary  output. 

Objectives  are  of  two  classes — achromatic  and  apo- 
chromatic.  Those  in  general  use  are  of  the  achromatic 
type,  and  they  fulfil  all  requirements  for  ordinary  work. 
Apochromatic  objectives  are  more  highly  corrected  for 
chromatic  and  spheric  aberration,  and  represent  the 
highest  type  of  microscope  lenses  produced.  They  are 
very  desirable  for  photomicrographic  and  research  work, 
but  for  routine  laboratory  work  do  not  offer  advantages 
commensurate  with  their  great  cost.  They  require  the 
use  of  special  "  compensating  "  eye-pieces. 

The  "  working  distance  "  of  an  objective  should  not 
be  confused  with  its  focal  distance.  The  former  term 
refers  to  the  distance  between  the  front  lens  of  the  ob- 
jective, when  it  is  in  focus,  and  the  cover-glass.     It  is 


24  INTRODUCTION 

always  less  than  the  focal  distance,  since  the  "  focal 
point  "  lies  somewhere  within  the  objective;  and  it 
varies  considerably  with  different  makes.  Long  working 
distance  is  a  very  desirable  feature. 

Objectives  are  "  corrected  "  for  use  under  certain 
fixed  conditions,  and  they  will  give  the  best  results  only 
when  used  under  the  conditions  for  which  corrected.  The 
most  important  corrections  are:  (a)  For  tube-length; 
(b)  for  thickness  of  cover-glass;  and  (c)  for  the  medium 
between  objective  and  cover-glass. 

{a)  The  tube-length  with  which  an  objective  is  to  be 
used  is  usually  engraved  upon  it — in  most  cases  it  is 
i6o  mm.  The  draw-tube  of  the  microscope  should  be 
pulled  out  until  the  proper  length  is  obtained,  as  indi- 
cated by  the  graduations  on  its  side.  When  a  nose-piece 
is  used,  it  adds  about  15  mm.  to  the  tube-length,  and  the 
draw-tube  must  be  pushed  in  for  that  distance. 

(b)  The  average  No.  2  cover-glass  is  about  the  thick- 
ness for  which  most  objectives  are  corrected — usually  0.17 
or  0.18  mm.  Very  low  powers  and  oil-immersion  objec- 
tives do  not  require  any  cover-glass.  A  cover  should 
always  be  used  with  high  dry  lenses,  but  its  exact  thick- 
ness is  more  important  in  theory  than  in  practice. 
Many  immersion  objectives  have  such  short  working 
distance  that  only  very  thin  covers  can  be  used. 

(c)  The  correction  for  the  medium  between  objective 
and  cover-glass  is  very  important.  This  medium  may  be 
either  air  or  sorrie  fluid,  and  the  objective  is  hence  either 
a  "  dry  "  or  an  "  immersion  "  objective.  The  immersion 
fluid  generally  used  is  cedar  oil,  which  gives  great  optical 
advantages  because  its  index  of  refraction  is  the  same  as 
that  of  crown  glass.     It  is  obvious  that  only  objectives 


USE   OF   THE   MICROSCOPE  25 

with  very  short  working  distance,  as  the  2  mm.,  can  be 
used  with  an  immersion  fluid. 

To  use  an  oil-immersion  objective  a  drop  of  the  cedar 
oil  which  is  prepared  for  the  purpose  should  be  placed 
upon  the  cover,  and  the  objective  lowered  into  it  and 
then  brought  to  a  focus  in  the  usual  way. 

Bubbles  in  the  oil  are  a  frequent  source  of  trouble, 
and  should  always  be  looked  for  when  an  immersion 
objective  does  poor  work.  They  are  readily  seen  by 
removing  the  eye-piece  and  looking  down  the  tube. 
Immediately  after  use  the  oil  should  be  removed  with 
lens-paper  or  a  soft  linen  handkerchief. 

A  useful  "  pointer  "  can  be  made  by  placing  a  straight 
piece  of  a  hair  across  the  opening  of  the  diaphragm  of 
the  eye-piece,  cementing  one  end  with  a  tiny  drop  of 
balsam,  and  cutting  the  hair  in  two  in  the  middle.  When 
the  eye-piece  is  in  place,  the  hair  appears  as  a  black  Une 
extending  from  the  periphery  to  the  center  of  the  micro- 
scopic field. 

Numeric  Aperture. — This  expression,  usually  written 
N.  A.,  indicates  the  amount  of  Ught  which  enters  an  ob- 
jective from  a  point  in  the  microscopic  field.  In  optical 
language,  N.  A.  is  the  sine  of  one-half  the  angle  of  aper- 
ture multiplied  by  the  index  of  refraction  of  the  medium 
between  the  cover  and  the  front  lens.  Numeric  aper- 
ture is  extremely  important,  because  upon  it  depends 
resolving  power,  which  is  the  most  important  property 
of  an  objective.' 

*  Resolving  power  really  depends  upon  two  factors,  the  N.  A.  and  the 
wave  length  of  light,  but  the  latter  can  be  ignored  in  practice.  The 
great  resolving  power  of  the  ultra-microscope  depends  upon  its  use  of 
light  of  short  wave  length. 


26  INTRODUCTION 

Resolving  power  is  the  ability  to  separate  minute 
details  of  structure.  For  example,  the  dark  portions  of 
a  good  half-tone  picture  appear  gray  or  black  to  the  un- 
aided eye,  but  a  lens  easily  resolves  this  apparently 
uniform  surface  into  a  series  of  separate  dots.  Resolv- 
ing power  does  not  depend  upon  magnification.  The 
fine  lines  and  dots  upon  certain  diatoms  may  be  brought 
out  clearly  and  crisply  {i.  e.,  they  are  resolved)  by  an 
objective  of  high  numeric  aperture,  whereas  with  an 
objective  of  lower  numeric  aperture,  but  greater  magnify- 
ing power,  the  same  diatom  may  appear  to  have  a  smooth 
surface,  W'ith  no  markings  at  all,  no  matter  how  greatly 
it  is  magnified.  Knowing  the  N.  A.,  it  is  possible  to 
calculate  how  closely  lines  and  dots  may  lie  and  still 
be  resolved  by  a  given  objective.  To  state  the  numeric 
aperture,  therefore,  is  to  tell  what  the  objective  can 
accomplish;  provided,  of  course,  that  spheric  and  chro- 
matic aberrations  are  satisfactorily  corrected.  An  ob- 
jective's N.  A.  is  usually  engraved  upon  the  mounting. 

It  is  an  important  fact,  and  one  almost  universally 
overlooked  by  practical  microscopists,  that  the  pro- 
portion of  the  numeric  aperture  of  an  objective  which  is 
utilized  depends  upon  the  aperture  of  the  cone  of  light 
delivered  by  the  condenser.  In  practice,  the  numeric 
aperture  of  an  objective  is  reduced  nearly  to  that  of 
the  condenser  (which  is  indicated  by  lower-case  letters, 
n.  a.).'  The  condenser  should,  therefore,  have  a 
numeric  aperture  at  least  equal  to  that  of  the  objective 
with  which  it  is  to  be  used.     Lowering  the  condenser 

'  The  N.  A.  of  the  objective  is  not  reduced  wholly  to  that  of  the  con- 
denser, because,  owing  to  difraction  phenomena,  a  small  part  of  the  un- 
illuminated  portion  of  the  black  lens  is  utilized. 


USE   OF   THE   MICROSCOPE  27 

below  its  focal  distance  and  closing  the  diaphragm  be- 
neath it  have  the  eflfect  of  reducing  its  working  aperture. 
A  condenser,  whatever  its  numeric  aperture,  cannot 
deliver  through  the  air  a  cone  of  light  of  greater  n.  a. 
than  I.  It  follows,  therefore,  that  the  proper  adjust- 
ment of  the  substage  condenser  is  a  matter  of  great  im- 
portance when  using  objectives  of  high  N.  A.,  and  that, 
to  gain  the  full  benefit  of  the  resolving  power  of  such 
objectives,  the  condenser  must  be  focused  on  the  object 
under  examination,  it  must  be  oiled  to  the  under  surface 
of  the  slide  in  the  same  way  as  the  immersion  objective 
is  oiled  to  the  cover-glass,  and  the  substage  diaphragm 
must  be  wide  open.  The  last  condition  introduces 
a  difficulty  in  that  colorless  structures  will  appear 
"fogged"  in  a  glare  of  light  (Fig.  4).  Wright  suggests 
that  the  size  of  the  light  source  be  so  regulated  by  a 
diaphragm  that  its  image,  thrown  on  the  slide  by  the 
condenser,  coincides  with  the  real  field  of  the  objective, 
and  maintains  that  in  this  way  it  is  possible  to  reduce 
the  glare  of  light  and  to  dispel  the  fog  without  closing 
the  diaphragm  of  the  condenser. 

One  can  easily  determine  how  much  of  the  aperture 
of  an  objective  is  in  use  by  removing  the  eye-piece,  look- 
ing down  the  tube,  and  observing  what  proportion  of  the 
back  lens  of  the  objective  is  illuminated.  The  relation 
of  the  illuminated  central  portion  to  the  unilluminated 
peripheral  zone  indicates  the  proportion  of  the  numeric 
aperture  in  use.  The  effect  of  raising  and  lowering  the 
condenser  and  of  oiling  it  to  the  slide  can  thus  be  easily 
seen. 

Magnification. — The  degree  of  magnification  should 
always  be  expressed  in  diameters,  not  times,  which  is  a 


28  INTRODUCTION 

misleading  term.  The  former  refers  to  increase  of 
diameter;  the  latter,  to  increase  of  area.  The  compara- 
tively low  magnification  of  loo  diameters  is  the  same  as 
the  apparently  enormous  magnification  of  10,000  times. 
The  magnifying  power  of  a  lens  is  obtained  by  dividing 
250  mm.,  or  10  inches  (the  distance  of  normal  vision), 
by  the  focal  length  of  the  lens.  The  focal  length  of  an 
objective  is  approximately  twice  the  diameter  of  the 
front  lens.  Thus,  the  2  mm.  objective  gives  a  mag- 
nification of  125  diameters;  the  25  mm.  eye-piece  gives 
a  magnification  of  10  diameters,  and  is  usually  designated 
as  a  10  X  eye-piece.  When  an  objective  and  eye-piece 
are  used  together,  the  total  magnification  is  the  product 
of  the  two.  In  the  case  just  cited  the  total  magnifica- 
tion would  be  1250  diameters.  In  practice,  magnifi- 
cation can  be  increased  in  one  of  three  ways: 

(a)  Drawing  out  the  tube.  Since  the  increased  tube- 
length  interferes  with  spheric  correction,  it  should  be 
used  only  with  the  knowledge  that  an  imperfect  image 
will  result. 

(b)  Using  a  higher  power  objective.  As  a  rule,  this  is 
the  best  way,  because  resolving  power  is  also  increased; 
but  it  is  often  undesirable  because  of  the  shorter  working 
distance,  and  because  the  higher  objective  often  gives 
greater  magnification  than  is  desired,  or  cuts  down  the 
size  of  the  real  field  to  too  great  an  extent. 

(c)  Using  a  shorter  eye-piece.  This  is  the  simplest 
method.  It  has,  however,  certain  limitations.  When 
too  high  an  eye-piece  is  used,  there  results  a  hazy  image 
in  which  no  structural  detail  is  seen  clearly.  This  is 
called  "  empty  magnification,"  and  depends  upon  the 
fact  that  the  objective  has  not  sufl[icient  resolving  power 


USE  OF  THE  MICROSCOPE  29 

to  support  the  high  magnification.  The  extent  to  which 
magnification  can  be  satisfactorily  increased  by  eye- 
piecing  depends  wholly  upon  the  resolving  power  of  the 
objective,  and  consequently  upon  the  N.  A.  The  great- 
est total  or  combined  magnification  which  will  give  an 
absolutely  crisp  picture  is  found  by  multiplying  the  N.  A. 
of  an  objective  by  400.  The  greatest  magnification 
which  can  be  used  at  all  satisfactorily  is  1000  times  the 
N.  A.  For  example:  The  ordinary  2  mm.  objective  has 
a  N.  A.  of  1.30;  the  greatest  magnification  which  will 
give  an  absolutely  sharp  picture  is  520  diameters^  which 
is  obtained  approximately  by  using  a  4X  eye-piece. 
Higher  eye-pieces  can  be  used,  up  to  a  total  magnifica- 
tion of  1300  diameters  (10  X  eye-piece),  beyond  which 
the  image  becomes  wholly  unsatisfactory. 

Focusing. — It  is  always  best  to  "  focus  up,"  which 
saves  annoyance  and  probable  damage  to  slides  and  ob- 
jectives. This  is  accomplished  by  bringing  the  objec- 
tive nearer  the  slide  than  the  proper  focus,  and  then,  with 
the  eye  at  the  eye-piece,  turning  the  tube  up  tmtil  the 
object  is  clearly  seen.  The  fine  adjustment  should  he 
used  only  to  get  an  exact  focus  with  the  higher  power  ob- 
jectives after  the  instrument  is  in  approximate  focus. 
It  should  not  be  turned  more  than  one  revolution. 

There  will  be  less  fatigue  to  the  eyes  if  both  are  kept 
open  while  using  the  microscope,  and  if  no  effort  is  made 
to  see  objects  which  are  out  of  distinct  focus.  Fine 
focusing  should  be  done  with  the  fine  adjustment,  not 
with  the  eye.  An  experienced  microscopist  keeps  his 
fingers  almost  constantly  upon  one  or  other  of  the  focus- 
ing adjustments.  Greater  skill  in  recognizing  objects 
will  be  acquired  if  the  same  eye  be  always  used.     To  be 


3©  INTRODUCTION 

seen  most  clearly,  an  object  should  be  brought  to  the 
center  of  the  field. 

Care  of  the  Microscope. — The  microscope  is  a  deH- 
cate  instrument  and  should  be  handled  accordingly.  It 
is  so  heavy  that  one  is  apt  to  forget  that  parts  of  it  are 
fragile.  It  seems  unnecessary  to  say  that  when  there 
is  unusual  resistance  to  any  manipulation,  force  should 
never  be  used  to  overcome  it  until  its  cause  has  first 
been  sought;  and  yet  it  is  no  uncommon  thing  to  see 
students,  and  even  graduates,  push  a  high-power  objec- 
tive against  a  microscopic  preparation  with  such  force 
as  to  break  not  only  the  cover-glass,  but  even  a  heavy 
slide. 

It  is  most  convenient  to  carry  a  microscope  with  the 
fingers  grasping  the  pillar  and  the  arm  which  holds  the 
tube;  but  since  this  throws  a  strain  upon  the  fine  adjust- 
ment, it  is  safer  to  carry  it  by  the  base.  In  the  more 
recent  instruments  a  convenient  handle-arm  is  provided. 
To  bend  the  instrument  at  the  joint,  the  force  should 
be  applied  to  the  pillar  and  never  to  the  tube  or  the  stage. 

Lens  surfaces  which  have  been  exposed  to  dust  only 
should  be  cleaned  with  a  camel's-hair  brush.  Those 
which  are  exposed  to  finger-marks  should  be  cleaned  with 
lens  paper,  or  a  soft  linen  handkerchief  wet  with  saliva. 
Particles  of  dirt  which  are  seen  in  the  field  are  upon  the 
slide,  the  eye-piece,  or  the  condenser.  Their  location 
can  be  determined  by  moving  the  slide,  rotating  the  eye- 
piece, and  lowering  the  condenser.  When  the  image  is 
hazy,  the  objective  probably  needs  cleaning;  or  in  case  of 
an  oil-immersion  lens,  there  may  be  bubbles  in  the  oil. 

Oil  and  balsam  which  have  dried  upon  the  lenses  and 
resist  saliva  may  be  removed  with  alcohol  or  xylol;  but 


USE   OF  THE   MICROSCOPE  3! 

these  solvents  must  be  used  sparingly  and  carefully,  as 
there  is  danger  of  softening  the  cement.  Care  must  be 
taken  not  to  get  any  alcohol  upon  the  brass  parts,  as  it 
will  remove  the  lacquer.  Balsam  and  dried  oil  are  best 
removed  from  the  brass  parts  with  xylol. 

Choice  of  a  Microscope. — It  is  poor  economy  to  buy 
a  cheap  instrument. 

For  the  work  of  a  clinical  laboratory  the  microscope 
should  preferably  be  of  the  new  handle-arm  type,  and 
should  have  a  large  stage.  It  should  be  provided  with  a 
substage  condenser  (preferably  of  1.40  n.  a.),  three  or 
more  objectives,  and  two  or  more  eye-pieces. 

The  most  generally  useful  objectives  are:  16  mm., 
4  mm.,  and  2  mm.  oil  immersion.  The  4  mm.  objective 
may  be  obtained  with  N.  A.  of  0.65  or  0.85.  If  it  is  to 
be  used  for  blood-counting,  the  former  is  preferable,  since 
its  working  distance  is  sufficient  to  take  the  thick  cover 
of  the  Thoma-Zeiss  instrument.  For  coarse  objects  a 
32  mm.  objective  is  very  desirable.  The  eye-pieces 
most  frequently  used  are  4  X  and  8  X .  A  very  low  power 
(2X)  and  a  very  high  (18  X)  will  sometimes  be  found 
useful.  The  micrometer  eye-piece  is  almost  a  necessity. 
A  mechanical  stage,  preferably  of  the  attachable  type, 
is  almost  indispensable  for  blood  and  certain  other  work. 

A  first-class  microscope,  of  either  American  or  foreign 
make,  equipped  as  just  described,  will  cost  in  the  neigh- 
borhood of  a  hundred  dollars,  exclusive  of  the  mechanical 
stage. 

-  Measurement  of  Microscopic  Objects. — Of  the  several 
methods,  the  most  convenient  is  the  use  of  a  micrometer 
eye-piece.  In  its  simplest  form  this  is  similar  to  an 
ordinary  eye-piece,  but  has  within  it  a  glass  disc  upon 


32  INTRODUCTION 

which  is  ruled  a  graduated  scale.  When  this  eye-piece  is 
placed  in  the  tube  of  the  microscope,  the  ruled  lines  ap- 
pear in  the  microscopic  field,  and  the  size  of  an  object  is 
readily  determined  in  terms  of  the  divisions  of  this  scale. 
The  value  of  these  divisions  in  inches  or  millimeters 
manifestly  varies  with  different  magnifications.  Their 
value  must,  therefore,  be  determined  separately  for  each 
objective.  This  is  accomplished  through  use  of  a  stage 
micrometer — a  glass  slide  with  carefully  ruled  scale 
divided  into  hundredths  and  thousandths  of  an  inch,  or 
into  subdivisions  of  a  millimeter.  •  The  stage  micrometer 
is  placed  upon  the  stage  of  the  microscope  and  brought 
into  focus.  From  the  number  of  divisions  of  the  eye- 
piece scale  corresponding  to  each  division  of  the  stage 
micrometer  the  value  of  the  former  in  fractions  of  an 
inch  or  millimeter  is  easily  calculated.  The  counting 
slide  of  the  Thoma-Zeiss  hemocytometer  will  answer 
in  place  of  a  stage  micrometer,  the  lines  which  form  the 
sides  of  the  small  squares  being  one-twentieth  of  a  milli- 
meter apart.  Any  eye-piece  can  be  converted  into  a 
micrometer  eye-piece  by  placing  a  micrometer  disc — 
a  small  circular  glass  plate  with  ruled  scale — ruled  side 
down  upon  its  diaphragm. 

The  principal  microscopic  objects  which  are  measured 
clinically  are  animal  parasites  and  their  ova  and  abnor- 
mal blood-corpuscles.  The  metric  system  is  used  almost 
exclusively.  For  very  small  objects  o.ooi  mm.  has  been 
adopted  as  the  unit  of  measurement,  under  the  name 
micron.  It  is  represented  by  the  Greek  letter  y..  For 
larger  objects,  where  exact  measurement  is  not  essential, 
the  diameter  of  a  red  blood-corpuscle  (7  to  8  /u)  is  some- 
times taken  as  a  unit. 


USE   OF   THE  MICROSCOPE  33 

Tuttle  has  suggested  that  in  feces  and  other  examina- 
tions a  little  lycopodium  powder  be  mixed  with  the 
material.  The  granules  are  of  uniform  size — 30  (i  in 
diameter — and  are  easily  recognized  (Fig.  5).  They 
furnish  a  useful  standard  with  which  the  size  of  other 
structures  can  be  compared. 


Fig.  s- — Egg  of  Tsenia  saginata.    Lycopodium  granules  used  as  micrometer  (X  250) 
(photograph  by  the  author). 


Photoniicrography. — Very  satisfactory  pictures  of  mi- 
croscopic structures  can  be  made  by  any  one  with  simple 
apparatus. 

Any  camera  with  focusing  screen  or  a  Kodak  with 
plate  attachment  may  be  used.  It  is  best,  but  not  neces- 
sary, to  remove  the  photographic  lens.  The  camera  is 
placed  with  the  lens  (or  lens-opening,  if  the  lens  has  been 
removed)  looking  into  the  eye-piece  of  the  microscope, 
which  may  be  in  either  the  vertical  or  the  horizontal 
position.  One  can  easily  rig  up  a  standard  to  which  the 
camera  can  be  attached  in  the  proper  position  by  means 
of  a  tripod  screw.  A  light-tight  connection  can  be  made 
3 


34  INTRODUCTION 

of  a  cylinder  of  paper  or  a  cloth  sleeve  with  draw-strings. 
The  image  will  be  thrown  upon  the  ground-glass  focusing 
screen,  and  is  focused  by  means  of  the  fine  adjustment  of 
the  microscope.  The  degree  of  magnification  is  ascer- 
tained by  placing  the  ruled  plate  of  the  blood-counting 
instrument  upon  the  microscope  and  measuring  the 
image  on  the  screen.  The  desired  magnification  is 
obtained  by  changing  objectives  or  eye-pieces  or  length- 
ening the  camera-draw. 

Focusing  is  comparatively  easy  with  low  powers,  but 
when  using  an  oil-immersion  objective,  it  is  a  difficult 
problem  unless  the  source  of  light  be  very  brilliant.  If 
one  always  uses  the  same  length  of  camera  and  micro- 
scope tube,  a  good  plan  is  as  follows:  Ascertain  by  trial 
with  a  strong  light  how  far  the  fine  adjustment  screw 
must  be  turned  from  the  correct  eye  focus  to  bring  the 
image  into  sharp  focus  upon  the  ground-glass  screen. 
At  any  future  time  one  has  only  to  focus  accurately 
with  the  eye,  bring  the  camera  into  position,  and  turn  the 
fine  adjustment  the  required  distance  to  right  or  left. 

The  Hght  should  be  as  intense  as  possible,  in  order  to 
shorten  exposure,  but  any  light  that  is  satisfactory  for 
ordinary  microscopic  work  will  answer.  It  is  nearly 
always  necessary  to  insert  a  color  screen  between  the 
light  and  the  microscope.  Pieces  of  colored  window- 
glass  are  useful  for  this  purpose.  The  screen  should  have 
a  color  complementary  to  that  which  it  is  desired  to  bring 
out  strongly  in  the  photograph:  for  blue  structures,  a 
yellow  screen;  for  red  structures,  a  green  screen.  For 
the  average  stained  preparation,  a  picric-acid  yellow  or  a 
yellow  green  will  be  found  satisfactory. 

Very  fair  pictures  can  be  made  on  Kodak  film,  but 


USE   OF   THE   MICROSCOPE  35 

orthochromatic  plates  (of  which  Cramer's  "  Iso  "  and 
Seed's  "  Ortho  "  are  examples)  give  much  better  re- 
sults. The  length  of  exposure  depends  upon  so  many 
factors  that  it  can  be  determined  only  by  trial.  It 
will  probably  vary  from  a  few  seconds  to  fifteen  minutes. 
Plates  are  developed  in  the  usual  way,— the  tank  method 
yielding  most  uniform  and  satisfactory  results, — but  in 
order  to  secure  all  the  contrast  possible,  they  should  be 
considerably  overdeveloped. 


Fig.  6. — ^Leukemic  blood  (about  X  650).    Photograph  taken  with  a  iiodak,  as  described 

in  the  text. 


The  photograph  from  which  Fig.  6  was  made  was  taken 
with  a  Kodak  and  plate  attachment  on  an  "  Iso  "  plate, 
the  source  of  light  being  the  electric  lamp  and  condensing 
lens  illustrated  in  Fig.  2.  It  was  focused  by  the  method 
described  above.  The  screen  was  a  picric-acid  stained 
photographic  plate.  Exposure,  three  and  a  half  min- 
utes. The  picture  loses  considerable  detail  in  reproduc- 
tion. 


CHAPTER  I 

THE  SPUTUM 

Preliminary  Considerations. — Before  beginning  the 
study  of  the  sputum,  the  student  will  do  well  to  familiar- 
ize himself  with  the  structures  which  may  be  present 
in  the  normal  mouth,  and  which  frequently  appear  in  the 
sputum  as  contaminations.  Nasal  mucus  and  material 
obtained  by  scraping  the  tongue  and  about  the  teeth 
should  be  studied  as  described  for  unstained  sputum.  A 
drop  of  Lugol's  solution  should  then  be  placed  at  the 
edge  of  the  cover,  and,  as  it  runs  under,  the  effect  upon 
different  structures  noted.  Another  portion  should  be 
spread  upon  slides  or  covers  and  stained  by  some  simple 
stain  and  by  Gram's  method.  The  structures  likely  to 
be  encountered  are  epithelial  cells  of  columnar  and 
squamous  types,  leukocytes,  food-particles,  Lepto- 
thrix  huccalis,  and  great  numbers  of  saprophytic  bac- 
teria, frequently  including  spirochetes.  These  struc- 
tures are  described  later. 

The  morning  sputum  or  the  whole  amount  for  twenty- 
four  hours  should  be  collected  for  examination.  In 
beginning  tuberculosis  tubercle  bacilli  can  often  be  found 
in  that  first  coughed  up  in  the  morning  when  they  can- 
not be  detected  at  any  other  time  of  day.  Sometimes,  in 
these  early  cases,  there  are  only  a  few  mucopurulent  flakes 
which  contain  the  bacilH,  or  only  a  small  purulent  mass 
every  few  days,  and  these  may  easily  be  overlooked. 

36 


THE   SPUTUM  37 

Patients  should  be  instructed  to  rinse  the  mouth 
well  in  order  to  avoid  contamination  with  food-particles 
which  may  prove  confusing  in  the  examination,  and  to 
make  sure  that  the  sputum  comes  from  the  lungs  or 
bronchi  and  not  from  the  nose  and  nasopharynx.  Many 
persons  find  it  difficult  to  distinguish  between  the  two. 
It  is  always  desirable  that  the  material  be  raised  with  a 
distinct  expulsive  cough,  but  this  is  not  always  possible. 
Material  from  the  upper  air-passages  can  usually  be  iden- 
tified from  the  large  proportion  of  mucus  and  the  charac- 
ter of  the  epithelial  cells. 

As  a  receptacle  for  the  sputum,  a  clean,  wide-mouthed 
bottle  with  tightly  fitting  cork  may  be  used.  The  pa- 
tient must  be  particularly  cautioned  against  smearing 
any  of  it  upon  the  outside  of  the  bottle.  This  is  prob- 
ably the  chief  source  of  danger  to  those  who  examine 
sputum.  Disinfectants  should  not  be  added.  They 
so  alter  the  character  of  the  sputum  as  to  render  it 
unfit  for  satisfactory  examination. 

"^Tien  the  examination  is  begun,  the  material  should 
be  spread  out  in  a  thin  layer  in  a  Petri  dish,  or  between 
two  small  plates  of  glass,  Hke  photographic  plates. 
It  may  then  be  examined  with  the  naked  eye — best 
over  a  black  background — or  with  a  low  power  of  the 
microscope.  The  portions  most  suitable  for  further 
examination  may  thus  be  easily  selected.  This  macro- 
scopic examination  should  never  he  omitted. 

After  an  examination  the  sputum  must  be  destroyed 
by  heat  or  chemicals,  and  everything  which  has  come  in 
contact  with  it  must  be  sterilized .  The  utmost  care  must 
be  taken  not  to  allow  any  of  it  to  dry  and  become  dis- 
seminated through  the  air.     It  is  a  good  plan  to  con- 


38  THE   SPUTUM 

duct  the  examination  upon  a  large  newspaper,  which 
can  then  be  burned.  Contamination  of  the  work  table 
is  thus  avoided.  If  this  is  not  feasible,  the  table  should 
be  washed  off  with  10  per  cent,  lysol  solution,  and  allowed 
to  dry  slowly,  as  soon  as  the  sputum  work  is  finished. 

Examination  of  the  sputum  is  most  conveniently  con- 
sidered under  four  heads:  I.  Physical  examination. 
II.  Microscopic  examination.  III.  Chemic  examination. 
IV.  Characteristics  of  the  sputum  in  various  diseases. 

I.  PHYSICAL  EXAMINATION 

1.  Quantity. — The  quantity  expectorated  in  twenty- 
four  hours  varies  greatly.  It  may  be  so  slight  as  to  be 
overlooked  entirely  in  beginning  tuberculosis.  It  is 
usually  small  in  acute  bronchitis  and  lobar  pneumonia. 
It  may  be  very  large — sometimes  as  much  as  1000  c.c. — 
in  advanced  tuberculosis  with  large  cavities,  edema  of  the 
lung,  bronchiectasis,  and  following  rupture  of  an  abscess 
or  empyema.  It  is  desirable  to  obtain  a  general  idea  of 
the  quantity,  but  accurate  measurement  is  unnecessary. 

2.  Color. — Since  the  sputum  ordinarily  consists  of 
varying  proportions  of  mucus  and  pus,  it  may  vary  from 
a  colorless,  translucent  mucus  to  an  opaque,  whitish  or 
yellow,  purulent  mass.  A  yellowish  green  is  frequently 
seen  in  advanced  phthisis  and  chronic  bronchitis.  In 
jaundice,  in  caseous  pneumonia,  and  in  slowly  resolv- 
ing lobar  pneumonia  it  may  assume  a  bright  green  color, 
due  to  bile  or  altered  blood-pigment. 

A  red  color  usually  indicates  the  presence  of  blood. 
Bright  red  blood,  most  commonly  in  streaks,  is  strongly 
suggestive  of  phthisis.  It  may  be  noted  very  early  in  the 
disease.     A  rusty  red  sputum  is  the  rule  in  croupous 


PHYSICAL  EXAMINATION  39 

pneumonia,  and  was  at  one  time  considered  pathogno- 
monic of  the  disease.  "  Prune- juice  "  sputum  is  said  to 
be  characteristic  of  ''  drunkard's  pneumonia."  It  at 
least  indicates  a  dangerous  type  of  the  disease.  A 
brown  color,  due  to  altered  blood-pigment,  follows 
hemorrhages  from  the  lungs,  and  is  present,  to  greater  or 
less  degree,  in  chronic  passive  congestion  of  the  lung, 
which  is  most  frequently  due  to  a  heart  lesion. 

Gray  or  black  sputum  is  observed  among  those  who 
work  much  in  coal-dust,  and  is  occasionally  seen  in 
smokers  who  are  accustomed  to  "  inhale." 

3.  Consistence. — According  to  their  consistence,  sputa 
are  usually  classified  as  serous,  mucoid,  purulent,  sero- 
purulent,  mucopurulent,  etc.,  which  names  explain  them- 
selves. As  a  rule,  the  more  mucus  and  the  less  pus  and 
serum  a  sputum  contains,  the  more  tenacious  it  is. 

The  rusty  sputum  of  croupous  pneumonia  is  extremely 
tenacious,  so  that  the  vessel  in  which  it  is  contained  may 
be  inverted  without  spilling  it.  The  same  is  true  of  the 
almost  purely  mucoid  sputum  ("  sputum  crudum  ")  of 
beginning  acute  bronchitis,  and  of  that  which  follows 
an  attack  of  asthma.  A  purely  serous  sputum,  usually 
slightly  blood  tinged,  is  fairly  characteristic  of  edema  of 
the  lungs. 

4.  Dittrich's  Plugs. — While  these  bodies  sometimes 
appear  in  the  sputum,  they  are  more  frequently  ex- 
pectorated alone.  They  are  caseous  masses,  usually 
about  the  size  of  a  pin-head,  but  sometimes  reaching 
that  of  a  bean.  The  smaller  ones  are  yellow,  the  larger 
ones  gray.  When  crushed,  they  emit  a  foul  odor. 
Microscopically,  they  consist  of  granular  debris,  fat-glob- 
ules, fatty  acid  crystals,  and  bacteria.    They  are  formed 


40  THE   SPUTUM 

in  the  bronchi,  and  are  sometimes  expectorated  by 
healthy  persons,  but  are  more  frequent  in  putrid  bron- 
chitis and  bronchiectasis.  The  laity  commonly  regard 
them  as  evidence  of  tuberculosis.  The  similar  caseous 
masses  which  are  formed  in  the  crj'pts  of  the  tonsils  are 
sometimes  also  included  under  this  name. 

11.  MICROSCOPIC  EXAMINATION 

The  portions  most  likely  to  contain  structures  of 
interest  should  be  very  carefully  selected,  as  already 
described.  The  few  minutes  spent  in  this  preliminary 
examination  will  sometimes  save  hours  of  work  later. 
Opaque,  white  or  yellow  particles  are  most  frequently 
bits  of  food,  but  may  be  cheesy  masses  from  the  tonsils; 
small  cheesy  nodules,  derived  from  tuberculous  cavities 
and  containing  many  tubercle  bacilli  and  elastic  fibers; 
Curschmann's  spirals,  or  small  fibrinous  casts,  coiled  into 
little  balls;  or  shreds  of  mucus  with  great  numbers  of 
entangled  pus-corpuscles.  The  food-particles  most  apt 
to  cause  confusion  are  bits  of  bread,  which  can  be  recog- 
nized by  the  blue  color  which  they  assume  when  touched 
with  iodin  solution. 

Some  structures  are  best  identified  without  staining; 
others  require  that  the  sputum  be  stained. 

A.     Unstained  Sputum 

A  careful  study  of  the  unstained  sputum  should  be 
included  in  every  routine  examination.  It  best  reveals 
certain  structures  which  are  seen  imperfectly  or  not  at 
all  in  stained  preparations.  It  gives  a  general  idea  of 
the  other  structures  which  are  present,  such  as  pus- 


MICROSCOPIC  EXAMINATION  41 

corpu§cles,  eosinophiles,  epithelial  cells,  and  blood,  and 
thus  suggests  appropriate  stains  to  be  used  later. 

The  particle  selected  for  examination  should  be  trans- 
ferred to  a  clean  sUde,  covered  with  a  clean  cover-glass, 
and  examined  with  the  i6  mm.  objective,  followed  by 
the  4  mm.  It  is  convenient  to  handle  the  bits  of 
sputum  with  a  wooden  tooth-pick  or  with  a  wooden 
cotton-applicator,  which  may  be  burned  when  done  with. 
The  platinum  wire  used  in  bacteriologic  work  is  less 
satisfactory  because  not  usually  stiff  enough. 

The  more  important  structures  to  be  seen  in  unstained 
sputum  are:  elastic  fibers,  Curschmann's  spirals,  Char- 
cot-Leyden  crystals,  fibrinous  casts,  the  ray  fungus  of 
actinomycosis,  and  molds.  Pigmented  cells,  especially 
the  so-called  "  heart-failure  cells  "  (p.  62),  are  also  best 
studied  without  staining  (Plate  II,  Fig.  i). 

1.  Elastic  Fibers.— These  are  the  elastic  fibers  of 
the  pulmonary  substance  (Fig.  7).  When  found  in  the 
sputum,  they  always  indicate  destructive  disease  of  the 
lung,  provided  they  do  not  come  from  the  food,  which  is 
a  not  infrequent  source.  They  are  found  most  com- 
monly in  phthisis;  rarely  in  other  diseases.  Advanced 
cases  of  tuberculosis  often  show  great  numbers,  and, 
rarely,  they  may  be  found  in  early  tuberculosis  when  the 
bacilli  cannot  be  detected.  In  gangrene  of  the  lung, 
contrary  to  the  older  teaching,  elastic  tissue  is  probably 
always  present  in  the  sputum,  usually  in  large  fragments. 

The  fibers  should  be  searched  for  with  a  16  mm. 
objective,  although  a  higher  power  is  needed  to  identify 
them  with  certainty.  Under  the  4  mm.  they  appear  as 
slender,  highly  refractive  fibers  with  double  contour,  and 
often  curled  or  spHt  ends.      Frequently  they  are  found 


42  THE  SPUTUM 

in  alveolar  arrangement,  retaining  the  original  outline  of 
the  alveoli  of  the  lung  (Fig.  7,  6).  This  arrangement 
is  positive  proof  of  their  origin  in  the  lung.  Leptothrix 
buccalis,  which  is  a  normal  inhabitant  of  the  mouth, 
may  easily  be  mistaken  for  elastic  tissue.  It  can  be  dis- 
tinguished by  running  a  little  iodin  solution  under  the 
cover-glass  (see  p.  56). 


Fig.  7. — Elastic  fibers  from  the  sputum:  o,  Highly  magnified;  b,  alveolar  arrangement, 
less  highly  magnified  (after  Bizzozero). 


Fatty-acid  crystals,  which  are  often  present  in  Dit- 
trich's  plugs  and  in  sputum  which  has  lain  in  the  body 
for  some  time,  also  simulate  elastic  tissue  when  very 
long,  but  they  are  more  like  stiff,  straight  or  curved 
needles  than  wavy  threads.  They  show  varicosities 
when  the  cover-glass  is  pressed  upon.  The  structures 
which  most  frequently  confuse  the  student  are  the  cotton 
fibrils  which  are  present  as  a  contamination  in  most 


MICROSCOPIC  EXAMINATION  43 

sputa.  These  are  usually  coarser  than  elastic  fibers,  and 
flat,  with  one  or  two  twists,  and  often  have  longitudinal 
striations  and  frayed-out  ends. 

To  find  elastic  fibers  when  not  abundant,  boil  the 
sputum  with  a  10  per  cent,  solution  of  caustic  soda  until 
it  becomes  fluid;  add  several  times  its  bulk  of  water,  and 
centrifugalize,  or  allow  to  stand  for  twenty-four  hours  in 
a  conical  glass.  Examine  the  sediment  microscopically. 
The  fibers  will  be  pale  and  swollen  and,  therefore, 
somewhat  difficult  to  recognize.  Too  long  boiling  will 
destroy  them  entirely. 

The  above  procedure,  although  widely  recommended, 
will  rarely  or  never  be  necessary  if  the  sputum  is  care- 
fully examined  in  a  thin  layer  against  a  black  back- 
ground macroscopically  and  with  a  hand-lens,  and  if  all 
suspicious  portions  are  further  studied  with  the  micro- 
scope. 

2.  Curschmann's  Spirals.— These  pecuHar  structures 
are  found  most  frequently  in  bronchial  asthma,  of 
which  they  are  fairly  characteristic.  They  may  occa- 
sionally be  met  with  in  chronic  bronchitis  and  other 
conditions.  Their  nature  has  not  been  definitely  deter- 
mined. 

Macroscopically,  they  are  whitish  or  yellow,  twisted 
threads,  frequently  coiled  into  little  balls  (Fig.  8,  I). 
Their  length  is  rarely  over  half  an  inch,  though  it  some- 
times exceeds  two  inches.  Under  a  16  mm.  objective 
they  appear  as  mucous  threads  having  a  clear  central 
fiber,  about  which  are  wound  many  fine  fibrils  (Fig.  8, 
II.  and  III.).  Eosinophiles  are  usually  present  within 
them,  and  sometimes  Charcot-Leyden  crystals.  Not 
infrequently  the  spirals  are  imperfectly  formed,  con- 


44 


THE   SPUTUM 


sisting  merely  of  twisted  strands  of  mucus  inclosing 
leukocytes.     The  central  fiber  is  absent  from  these. 

3.  Charcot=Leyden  Crystals.— Of  the  crystals  which 
may  be  found  in  the  sputum,  the  most  interesting  are  the 
Charcot-Leyden  crystals.  They  may  be  absent  when 
the  sputum  is  expectorated,  and  appear  in  large  numbers 
after  it  has  stood  for  some  time.     They  are  rarely  found 


,M 


I.  II  III. 

Fig.  8. — Curschmann's  spirals:  I.,  Natural  size;  II.  and  HI.,  enlarged:  a,  central  fiber 
(after  Curschmann). 


except  in  cases  of  bronchial  asthma,  and  were  at  one 
time  thought  to  be  the  cause  of  the  disease.  They 
frequently  adhere  to  Curschmann  spirals.  Their  exact 
nature  is  unknown.  Their  formation  seems  to  be  in 
some  way  connected  with  the  presence  of  eosinophilic 
cells.  Outside  of  the  sputum  they  are  found  in  the 
feces  in  association  with  animal  parasites,  and  in  the 
coagulated  blood  in  leukemia. 


MICROSCOPIC  EXAMINATION 


45 


They  are  colorless,  pointed,  often  needle-like,  octa- 
hedral crystals  (Fig.  9).  Their  size  varies  greatly,  the 
average  length  being  about  three  or  four  times  the 
diameter  of  a  red  blood-corpuscle. 


Fig.  9. — Charcot-Leyden  crystals  (after  Riegel). 

Other  crystals — hematoidin,  cholesterin,  and,  most 
frequently,  fatty-acid  needles  (see  Fig.  36)^ — are  common 
in  sputum  which  has  remained  in  the  body  for  a  consider- 
able time,  as  in  abscess  of  the  lung  and  bronchiectasis. 

4.  Fibrinous  Casts.— These  are  casts  of  the  bronchi, 
frequently,  but  not  always,  composed  of  fibrin.  In 
color  they  are  usually  white  or  grayish,  but  may  be 
reddish  or  brown,  from  the  presence  of  blood-pigment. 
Their  size  varies  with  that  of  the  bronchi  in  which  they 
are  formed.  They  may,  rarely,  be  fifteen  or  more 
centimeters  in  length.    When  large,  they  can  be  recog- 


46  THE    SPUTUM 

nized  with  the  naked  eye  by  floating  them  out  in  water 
over  a  black  surface;  when  small,  a  low  power  of  the 
microscope  must  be  used.  Their  branching,  tree-like 
structure  (Fig.  10)  is  usuall}^  sufficient  to  identify  them. 
Fibrinous  casts  are  characteristic  of  fibrinous  bron- 
chitis, but  may  also  be  found  in  diphtheria  of  the  smaller 
bronchi.  Very  small  casts  are  often  seen  in  croupous 
pneumonia. 


Fig.  10. — Fibrinous  bronchial  cast  (Sahli). 

5.  Actinomyces  Bovis  (Ray=fungus). — In  the  sputum 
of  pulmonary  actinomycosis  and  in  the  pus  from  actino- 
mycotic lesions  elsewhere  small,  yellowish,  "  sulphur  " 
granules  can  be  detected  with  the  unaided  eye.  With- 
out a  careful  macroscopic  examination  they  are  almost 
certain  to  be  overlooked.  The  fungus  can  be  seen  by 
crushing  one  of  these  granules  between  slide  and  cover, 
and  examining  with  a  low  power.     It  consists  of  a  net- 


MICROSCOPIC  EXAMINATION 


47 


work  of  threads  having  a  more  or  less  radial  arrangement, 
those  at  the  periphery  presenting  club-shaped  extremi- 
ties (Fig.  ii).  It  can  be  brought  out  more  clearly  by 
running  a  little  solution  of  eosin  in  alcohol  and  glycerin 
under  the  cover.  This  organism,  also  called  Strepto- 
thrix  actinomyces,  apparently  stands  midway  between 
the  bacteria  and  the  molds.  It  stains  by  Gram's  method. 
Actinomycosis  of  the  lung  is  rare.  The  clinical  pic- 
ture is  that  of  tuberculosis. 


Fig.  n. — Sputum  from  a  case  of  actinomycosis;  stained  (Jakob). 

6.  Molds  and  Yeasts.— The  hyphae  and  spores  of 
various  molds  are  occasionally  met  with  in  the  sputum. 
They  are  usually  the  result  of  contamination,  and  have 
little  significance.  The  hyphae  are  rods,  usually  jointed 
or  branched  (Fig.  62),  and  often  arranged  in  a  mesh  work 
(myceHum);  the  spores  are  highly  refractive  spheres. 
Both  stain  well  with  the  ordinary  stains. 

In  the  extremely  rare  condition  of  systemic  blasto- 
mycosis the  specific  yeasts  have  been  found  in  the  sputum 


48  THE    SPUTUM 

in  large  numbers.     It  is  advisable  to  add  a  little  lo  per 
cent,  caustic  soda  solution  and  examine  unstained. 

7.  Animal  Parasites.— These  are  extremely  rare  in 
the  sputum  in  this  country.  A  trichomonad,  perhaps 
identical  with  Trichomonas  vaginalis,  has  been  seen  in 
the  sputum  of  putrid  bronchitis  and  gangrene  of  the 
lung,  but  its  causal  relationship  is  doubtful.  In  Japan, 
infection  with  the  lung  flukeworm,  Paragonimus  wes- 
termani,  is  common,  and  the  ova  are  found  in  the 
sputum.  The  lung  is  not  an  uncommon  seat  for  echino- 
coccus  cysts,  and  booklets  and  scolices  may  appear,  as 
may  also  A  moeba  histolytica,  when  a  hepatic  abscess  has 
ruptured  into  the  lung.  Ciliated  body-cells,  with  cilia 
in  active  motion,  are  not  infrequently  seen,  and  may 
easily  be  mistaken  for  infusoria.  All  the  above-men- 
tioned parasites  are  described  in  Chapter  VI. 

B.  Stained  Sputum 

Structures  which  are  best  seen  in  stained  sputum  are 
bacteria  and  cells. 

A  number  of  smears  should  be  made  upon  slides  or 
covers,  dried  in  the  air,  and  fixed  in  the  flame,  as  de- 
scribed on  the  next  page.  Fixation  will  kill  the  bac- 
teria when  covers  are  used,  and  the  smears  may  be  kept 
indefinitely;  but  smears  on  slides  are  often  not  sterile, 
and  should  be  handled  accordingly.  One  of  the  smears 
should  be  stained  with  some  simple  stain,  like  Lofiler's 
methylene-blue,  which  will  give  a  good  idea  of  the 
various  cells  and  bacteria  present.  Special  stains  may 
then  be  applied,  as  indicated,  but  a  routine  examination 
should,  in  all  cases,  include  a  stain  by  the  method  for 
the  tubercle  bacillus  and  by  Gram's  method. 


MICROSCOPIC  EXAMINATION  49 

1 .  Bacteria. — Saprophytic  bacteria  from  mouth  con- 
tamination are  frequently  present  in  large  numbers  and 
will  prove  confusing  to  the  inexperienced.  The  pres- 
ence of  squamous  cells  in  their  neighborhood  will  sug- 
gest their  source.  Among  the  pathogenic  organisms 
which  have  clinical  importance  are:  tubercle  bacilli; 
staphylococci  and  streptococci;  pneumococci;  bacilli  of 
Friedlander;  influenza  bacilU,  and  Micrococcus  catar- 
rhdlis. 

(i)  Tubercle  Bacillus. — The  presence  of  the  tubercle 
bacillus  may  be  taken  as  positive  evidence  of  the  ex- 
istence of  tuberculosis  somewhere  along  the  respiratory 
tract,  most  likely  in  the  lung.  In  laryngeal  tuberculosis 
it  is  not  easily  found  in  the  sputum,  but  can  fre- 
quently be  detected  in  swabs  made  directly  from  the 
larynx. 

Recognition  of  the  tubercle  bacillus  depends  upon  the 
fact  that  it  stains  with  difficulty;  but  that  when  once 
stained,  it  retains  the  stain  tenaciously,  even  when 
treated  with  a  mineral  acid,  which  quickly  removes  the 
stain  from  other  bacteria.  This  "  acid-fast  "  property 
is  due  to  the  presence  of  a  waxy  capsule.  The  most 
convenient  method  for  general  purposes  is  here  given 
in  detail: 

Gabbet's  Method. — (i)  Spread  suspicious  particles  thinly 
and  evenly  upon  a  slide  or  a  cover-glass  held  in  the  grasp  of 
cover-glass  forceps.  In  general,  slides  are  more  satisfactory, 
but  cover-glasses  are  easier  to  handle  while  staining.  Do  not 
grasp  a  cover  too  near  the  edge  or  the  stain  will  not  stay 
on  it  well.  Tenacious  sputum  will  spread  better  if  gently 
warmed  while  spreading. 

(2)  Dry  the  film  in  the  air. 
4 


50  THE   SPUTUM 

(3)  Fix  in  a  flame;  i.  e.,  pass  the  cover-glass  rather  slowly, 
with  film  side  up,  three  times  (a  slide  about  twelve  times) 
through  the  flame  of  a  Bunsen  burner  or  alcohol  lamp  low 
down  in  the  flame.  Take  care  not  to  scorch.  Should  the 
film  be  washed  off  during  future  manipulations,  fixation  has 
been  insufficient. 

(4)  Apply  as  much  carbolfuchsin  as  will  stay  on,  and  hold 
over  a  flame  so  that  it  will  steam  for  three  minutes  or  longer, 
replacing  the  stain  as  it  evaporates.  If  the  bacilli  are  well 
stained  in  this  step,  there  will  be  little  danger  of  decolorizing 
them  later.  Too  great  heat  will  interfere  with  the  staining 
of  some  of  the  bacilli,  probably  by  destroying  the  waxy 
envelop  upon  which  the  acid-fast  property  depends.  It  is 
better  to  stain  at  room  temperature  for  twelve  to  twenty- 
four  hours. 

(5)  Wash  the  film  in  water. 

(6)  Apply  Gabbet's  stain  to  the  under  side  of  the  cover- 
glass  to  remove  excess  of  carbolfuchsin,  and  then  to  the  film 
side.     Allow  this  to  act  for  one-fourth  to  one-half  minute. 

(7)  Wash  in  water. 

(8)  If,  now,  the  thinner  portions  of  the  film  are  blue,  pro- 
ceed to  the  next  step;  if  they  are  still  red,  repeat  steps  (6) 
and  (7)  until  the  red  has  disappeared.  Too  long  application 
of  Gabbet's  stain  will  decolorize  the  tubercle  bacilli. 

(9)  Place  the  preparation  between  layers  of  filter-paper  and 
dry  by  rubbing  with  the  fingers,  as  one  would  in  blotting  ink, 

(10)  Put  a  drop  of  Canada  balsam  upon  a  clean  slide,  place 
the  cover-glass  film  side  down  upon  it,  and  examine  with  an 
immersion  objective.  Cedar  oil  or  water  may  be  used  in 
place  of  balsam  for  temporary  preparations.  Smears  on 
slides  may  be  examined  directly  with  an  oil-immersion  lens, 
no  cover  being  necessary. 

Carbolfuchsin  is  prepared  by  mixing  10  c.c.  of  a  saturated 
alcoholic  solution  of  fuchsin  with  90  c.c.  of  5  per  cent,  aqueous 
solution  of  phenol. 


MICROSCOPIC  EXAMINATLON  CI 

Gabbet's  stain  consists  of  n^^^ylene-bIue/0  2  §nt\,;^25  per 
cent,  sulphuric  acid,  loo  c.c.  '^^^-l/>  fir    ^       '  ^^^f^fiTrj 

Both  stains  can  be  purchased  ready  prepared;    -'^  Up  f-_^ 

Other  Methods. — The  objection  is  often  raised  that  de-  'v /^ 
colorization  is  masked  by  the  blue  in  Gabbet's  stain,  but  this 
will  not  make  trouble  if  step  8  is  carefully  carried  out.  The 
Ziehl-Neelsen  method  is  preferred  by  many:  After  the  stain- 
ing with  carbolfuchsin  the  smear  is  washed  in  5  per  cent, 
nitric  acid  until  decolorized,  washed  in  water,  stained  lightly 
with  LofHer's  methylene-blue,  again  washed,  and  mounted. 

Pappenheim's  Method. — This  is  the  same  as  Gabbet's 
method,  except  that  Pappenheim's  methylene-blue  solution 
is  substituted  for  Gabbet's  solution.    This  consists  of: 

Corallin  (rosolic  acid) i  gm. 

Absolute  alcohol 100  c.c. 

Saturate  with  methylene-blue  and  add  20  c.c.  glycerin. 

The  method  is  very  satisfactory  for  routine  work.  De- 
colorization  of  the  tubercle  bacillus  is  practically  impos- 
sible: it  retains  its  red  color,  even  when  soaked  overnight 
in  Pappenheim's  solution.  The  stain  was  originally  recom- 
mended as  a  means  of  differentiating  the  smegma  bacillus, 
which  is  decolorized  by  it;  but  it  is  not  to  be  absolutely  relied 
upon  for  this  purpose. 

In  films  stained  by  these  methods  tubercle  bacilli, 
if  present,  will  be  seen  as  slender  red  rods  upon  a  blue 
background  of  mucus  and  cells  (Plate.  II,  Fig.  2).  They 
average  3  to  4  ll  in  length — about  one-half  the  diameter 
of  a  red  blood-corpuscle.  Beginners  must  be  warned 
against  mistaking  the  edges  of  cells,  or  particles  which 
have  retained  the  red  stain,  for  bacilli.  The  appear- 
ance of  the  bacilli  is  almost  always  typical,  and  if  there 


52  THE    SPUTUM 

seems  room  for  doubt,  the  structure  in  question  is  prob- 
ably not  a  tubercJe  bacillus.  They  may  lie  singly  or  in 
groujw.  They  are  very  frequently  bent  and  often  have 
a  beaded  appearance.  It  is  possible  that  the  larger, 
beaded  bacilli  indicate  a  less  active  tuberculous  process 
than  do  the  smaller,  uniformly  stained  ones.  Some- 
times they  are  present  in  great  numbers — thousands 
in  a  field  of  the  2  mm.  objective.  Sometimes  sev- 
eral cover-glasses  must  be  examined  to  find  a  single 
bacillus.  At  times  they  are  so  few  that  none  are  found 
in  stained  smears,  and  special  methods  are  required 
to  detect  them.  The  number  may  bear  some  relation 
to  the  severity  of  the  disease,  but  this  relation  is  by  no 
means  constant.  The  mucoid  sputum  from  an  incip- 
ient case  sometimes  contains  great  numbers,  while 
sputum  from  large  tuberculous  cavities  at  times  contains 
very  few.  Failure  to  find  them  is  not  conclusive, 
though  their  absence  is  much  more  significant  when  the 
sputum  is  purulent  than  when  it  is  mucoid. 

When  they  are  not  found  in  suspicious  cases,  one  of 
the  following  methods  should  be  tried: 

(i)  Antiformin  Method. — This  has  lately  come  into  use, 
and  has  superseded  the  older  methods  of  concentration. 
The  chief  difficulty  with  the  older  methods,  such  as  boiling 
with  caustic  soda,  is  that  the  bacilli  are  so  injured  in  the 
process  that  they  do  not  stain  characteristically. 

Antiformin  is  the  patented  name  for  a  preparation  con- 
sisting essentially  of  equal  parts  of  a  15  per  cent,  solution  of 
caustic  soda  and  a  20  per  cent,  solution  of  sodium  hypo- 
chlorite. It  keeps  fairly  well.  The  sputum  is  thoroughly 
shaken  in  a  corked  bottle  with  one-fourth  its  volume  of  anti- 
formin, and  allowed  to  stand  four  to  six  hours  in  an  incubator, 


PLATE  II 


Fig.   I. — Heart-failure  cells  in  sputum,  containing  blood-pigment,  from 
a  case  of  cardiac  congestion  of  the  lungs  (Jakob). 


Fig.  2. — A,  Sputum  showing  tubercle  bacilli  stained  with  car- 
bolfuchsin  and  Gabbet's  methylene-blue  solution  (obj.  one-twelfth 
oil-immersion);  B,  sputum  of  anthracosis,  showing  particles  of  coal-dust 
stained  with  methylene-blue  (obj.  one-twelfth  oil-immersion)  (Boston). 


MICROSCOPIC  EXAMINATION  53 

or  twenty-four  hours  at  room-temperature.  The  sputum  will 
be  thoroughly  liquefied.  A  centrifuge  tubeful  is  thoroughly 
centrifugalized,  the  supernatant  fluid  is  poured  off  and 
replaced  with  water,  and  centrifugalization  is  repeated. 
This  washing  is  repeated  several  times.  Some  of  the  sedi- 
ment is  then  spread  upon  slides  (with  a  little  egg-albumen 
or  some  of  the  untreated  sputum  to  cause  it  to  adhere), 
dried,  fixed,  and  stained.  The  tubercle  bacilli  are  not  killed, 
but  retain  their  form  and  staining  properties  unchanged. 
Other  bacteria  and  cells  are  destroyed. 

Since  the  bacilli  remain  alive,  the  utmost  care  must  be 
used  in  handling,  and  all  tubes  and  glassware  which  have 
come  in  contact  with  the  liquefied  sputum  must  be 
sterilized. 

(2)  Animal  Inoculation. — Inoculation  of  guinea-pigs  is 
the  court  of  last  appeal  in  detection  of  tubercle  bacilli.  The 
method  is  described  on  p.  375-- 

There  are  a  number  of  bacilli,  called  acid-fast  bacilli, 
which  stain  in  the  same  way  as  the  tubercle  bacillus. 
They  stain  with  difficulty,  and  when  once  stained,  retain 
the  color  even  when  treated  with  a  mineral  acid;  but, 
unlike  the  tubercle  bacillus,  most  of  them  can  be  decolor- 
ized with  alcohol.  Of  these,  the  smegma  bacillus  is  the 
only  one  likely  ever  to  cause  confusion.  It,  or  a  similar 
bacillus,  is  sometimes  found  in  the  sputum  of  gangrene 
of  the  lung.  It  occurs  normally  about  the  glans  penis 
and  the  clitoris,  and  is  often  present  in  the  urine  and  in 
the  wax  of  the  ear.  The  method  of  distinguishing  it 
from"  the  tubercle  bacillus  is  given  later  (p.  168). 

Other  bacteria  than  the  acid-fast  group  are  stained 
blue  by  Gabbet's  method.  Those  most  commonly  found 
are  staphylococci,  streptococci,  and  pneumococci.  Their 


54  THE   SPUTUM 

presence  in  company  with  the  tubercle  bacillus  consti- 
tutes mixed  infection,  which  is  much  more  serious  than 
single  infection  by  the  tubercle  bacillus.  It  is  to  be 
remembered,  however,  that  a  few  of  these  bacteria  may 
reach  the  sputum  from  the  upper  air-passages.  Clini- 
cally, mixed  infection  is  evidenced  by  fever. 

(2)  Staphylococcus  and  Streptococcus  (p.  368). — One 
or  both  of  these  organisms  is  commonly  present  in  com- 
pany with  the  tubercle  bacillus  in  the  sputum  of  ad- 
vanced phthisis  (Plate  II,  Fig.  2).  They  are  often 
found  in  bronchitis,  catarrhal  pneumonia,  and  many 
other  conditions. 

(3)  Pneumococcus  (Diplococcus  of  Frankel). — ^The 
pneumococcus  is  the  causative  agent  in  nearly  all  cases 
of  croupous  pneumonia,  and  is  commonly  found  in  large 
numbers  in  the  rusty  sputum  of  this  disease.  It  is  some- 
times met  with  in  the  sputum  of  catarrhal  pneumonia, 
bronchitis,  and  tuberculosis.  It  is  also  an  important 
factor  in  the  causation  of  pleurisy,  meningitis,  otitis 
media,  and  other  inflammations.  It  has  been  found  in 
the  saliva  in  health.  Pneumococci  are  about  the  size  of 
streptococci.  They  are  ovoid  in  shape,  and  lie  in  pairs, 
end  to  end,  often  forming  short  chains.  Each  is  sur- 
rounded by  a  gelatinous  capsule,  which  is  its  distinctive 
feature  (Fig.  12).  Diplococci  without  capsules  are  com- 
mon in  the  sputum,  but  have  no  special  significance. 

The  pneumococcus  is  closely  related  to  the  strepto- 
coccus, and  it  is  sometimes  extremely  difficult  to  differ- 
entiate them  even  by  culture  methods  (for  which  see 
p.  368).  The  morphology  of  the  pneumococcus,  the 
fact  that  it  is  Gram-positive,  and  the  presence  of  a  cap- 
sule are,  however,  generally  sufficient  for  its  recognition 


MICROSCOPIC   EXAMINATION  55 

in  smears  from  sputum  or  pus.  The  capsule  is  often 
seen  as  a  halo  around  pairs  of  cocci  in  smears  stained 
by  the  ordinary  methods,  particularly  Gram's  method, 
but  to  show  it  well  special  methods  are  required. 
There  are  numerous  special  methods  of  staining  cap- 
sules which  are  applicable  to  other  encapsulated  bacteria, 
as  well  as  to  the  pneumococcus,  but  few  of  them  are 
satisfactory.  Buerger's  method  can  be  recommended. 
It  is  especially  useful  with  cultures  upon  serum  media, 


Fig.  12. — Diplococcus  pneumonix  in  the  blood  (  X  looo)  (Frankel  and  Pfeiffer). 

but  is  applicable  also  to  the  sputum.  Smith's  new 
method  is  easier  of  application,  and  apparently  gives 
uniformly  good  results.  The  India-ink  method  described 
for  the  organism  of  syphilis  is  likewise  said  to  show 
capsules  satisfactorily.  The  sputum  should  be  fresh — 
not  more  than  three  or  four  hours  old. 

Buerger's  Method  for  Capsules. — (i)  Mix  a  few  drops 
each  of  the  sputum  and  blood-serum  or  egg-albumen  solu- 


56  THE    SPUTUM 

tion  (egg-albumen,  distilled  water,  equal  parts;  shake  and 
filter  through  cotton).  Blood-serum  can  be  obtained  as 
described  for  the  Widal  test,  p.  258.  Make  thin  smears  from 
the  mixture,  and  just  as  the  edges  begin  to  dry,  cover  with 
Miiller's  fluid  (potassium  dichromate,  2.5  gm.;  sodium  sul- 
phate, i.o  gm.;  water,  100  c.c.)  saturated  with  mercuric 
chlorid  (ordinarily  about  5  per  cent.).  Gently  warm  over 
a  flame  for  about  three  seconds. 

(2)  Rinse  very  quickly  in  water, 

(3)  Flush  once  with  alcohol. 

(4)  Apply  tincture  of  iodin  for  one  to  two  minutes. 

(5)  Thoroughly  wash  off  the  iodin  with  alcohol  and  dry  in 
the  air. 

(6)  Stain  about  three  seconds  with  weak  anilin-gentian- 
violet  freshly  made  up  as  follows:  Anilin  oil,  10;  water,  100; 
shake;  filter;  and  add  5  c.c.  of  a  saturated  alcoholic  solution 
of  gentian  violet. 

(7)  Rinse  off  the  stain  with  2  per  cent,  solution  of  sodium 
chlorid,  mount  in  this  solution,  and  examine  with  a  one- 
twelfth  objective. 

Buerger  suggests  a  very  useful  variation  as  follows:  After 
the  alcohol  wash  and  drying,  the  specimen  is  stained  by 
Gram's  method  (p.  409),  counterstained  with  aqueous  solu- 
tion of  fuchsin,  washed,  and  mounted  in  water.  The 
pneumococcus  holds  the  purple  stain,  while  all  capsules 
take  on  the  pink  counterstain. 

Smith's  Method. — (i)  Make  thin  smears  of  the  sputum 
or  other  material,  which  should  be  as  fresh  as  possible. 

(2)  Fix  in  the  flame  in  the  usual  manner. 

(3)  Apply  a  10  per  cent,  aqueous  solution  of  phospho- 
molybdic  acid  (Merck)  for  four  to  five  seconds. 

(4)  Rinse  in  water. 

(5)  Apply  anilin-gentian- violet,  steaming  gently  for 
fifteen  to  thirty  seconds. 

(6)  Rinse  in  water. 


MICROSCOPIC  EXAMINATION  57 

(7)  Apply  Gram's  iodin  solution,  steaming  gently  for 
fifteen  to  thirty  seconds. 

(8)  Wash  in  95  per  cent,  alcohol  until  the  purple  color 
ceases  to  come  off. 

(9)  Rinse  in  water. 

(10)  Apply  a  6  per  cent,  aqueous  solution  of  eosin  (Grii- 
bler,  w.  g.),  and  gently  warm  for  one-half  to  one  minute. 

(11)  Rinse  in  water. 

(12)  Wash  in  absolute  alcohol. 

(13)  Clear  in  xylol. 

(14)  Mount  in  balsam. 

This  is  essentially  Gram's  method  (seep.  409),  preceded 
by  treatment  with  phosphomolybdic  acid  and  followed  by 
eosin.  Gram-positive  bacteria  like  the  pneumococcus  are 
deep  purple;  capsules  are  pink  and  stand  out  clearly. 

When  the  method  is  applied  to  Gram-negative  bacteria, 
steps  5  to  9  inclusive  are  omitted ;  between  steps  1 1  and  1 2 
the  preparation  is  counterstained  with  Loffler's  methylene- 
blue,  gently  warming  for  fifteen  to  thirty  seconds. 

A  nilin- gentian-violet. — Ehrlich's  formula  is  the  one  gener- 
ally used,  but  this  keeps  only  a  few  weeks.  Stirling's  solu- 
tion, which  keeps  much  better  and  seems  to  give  equal  results, 
is  as  follows:  gentian-violet,  5  gm.;  alcohol,  10  c.c;  anilin 
oil,  2  c.c;  water,  88  c.c. 

Formalin- gentian-violet  is  a  satisfactory  substitute  for 
anilin-gentian-violet  and  is  permanent.  It  consists  of  5  per 
cent,  solution  formalin,  75  parts;  saturated  alcohohc  solution 
gentian-violet,  25  parts. 

Gram's  Iodin  Solution. — Iodin,  i  gm.;  potassium  iodid, 
2  gm.;  water,  300  c.c. 

Loffler^s  alkaline  methylene-bliie  is  a  very  generally  useful 
stain  for  bacteria.  It  is  composed  of  30  parts  of  a  saturated 
alcoholic  solution  of  methylene-blue  and  100  parts  of  a 
I  :  10,000  aqueous  solution  of  caustic  potash.  It  keeps 
indefinitely. 


58  THE    SPUTUM 

(4)  Bacillus  of  Friedlander  (Bacillus  Mucosus  Cap- 
sulatus). — In  a  small  percentage  of  cases  of  pneumonia 
this  organism  is  found  alone  or  in  company  with  the 
pneumococcus.  Its  pathologic  significance  is  uncer- 
tain. It  is  often  present  in  the  respiratory  tract  under 
normal  conditions.  Friedliinder's  bacilli  are  non-motile, 
encapsulated  rods,  sometimes  arranged  in  short  chains 
(Fig.  13) .  Very  short  individuals  in  pairs  closely  resemble 
pneumococci,  from  which  they  are  distinguished  by  the 
fact  that  they  are  Gram-decolorizing. 

(5)  Bacillus  of  Influenza. — This 
is  the  etiologic  factor  in  true  in- 
fluenza, although  conditions  which 
are  clinically  similar  or  identical 
may  be  caused  by  the  pneumococ- 
cus, streptococcus,  or  Micrococcus 
catarrhalis.  It  is  present,  often  in 
Friediandcr's     large  uumbcrs,   in   the  nasal  and 

bacillus  in  pus  from  pulmon-  ,  ,  , 

ary  abscess  (obj.  one-twelfth)  brouchial  secrctions,  and  is  also 
(Boston).  found  in  the  local  lesions  following 

influenza.  Chronic  infection  by  influenza  bacilli  may 
be  mistaken  clinically  for  tuberculosis,  and  they  should 
be  searched  for  in  all  cases  of  obstinate  chronic  bron- 
chitis. 

Their  recognition  depends  upon  the  facts  that  they 
are  extremely  small  bacilli ;  that  most  of  them  lie  within 
the  pus-cells;  that  their  ends  stain  more  deeply  than  their 
centers,  sometimes  giving  the  appearance  of  minute 
diplococci;  and  that  they  are  decolorized  by  Gram's 
method  of  staining  (Figs.  14  and  149). 

They  are  stained  blue  in  the  methods  for  tubercle 
bacilli,  but  are  more  certainly  recognized   by  Gram's 


MICROSCOPIC  EXAMINATION  59 

method,   followed    by  a    counterstain.     Pappenheim's 
pyronin-methyl-green  stain  is  especially  satisfactory. 

(6)  Micrococcus  Catarrhalis. — This  organism  is  fre- 
quently present  in  the  sputum  in  inflammatory  condi- 
tions of  the  respiratory  tract  resembling  influenza. 
It  is  sometimes  present  in  the  nasal  secretions  in  health. 
It  is  a  Gram-negative  diplococcus,  frequently  intra- 
cellular, and  can  be  distinguished  from  the  meningo- 


Fig.  14. — Bacillus  of  influenza;  cover-glass  preparation  of  sputum  from  a  case  of  influenza, 
showing  the  bacilli  in  leukocytes;  highly  magnified  (Pfeiffer). 


coccus  and  gonococcus  only  by  means  of  cultures.  It 
grows  readily  on  ordinary  media. 

2.  Cells. — These  include  pus-corpuscles,  epithelial 
cells,  and  red  blood-corpuscles. 

(i)  Pus-corpuscles  are  present  in  every  sputum,  and 
at  times  the  sputum  may  consist  of  little  else.  They  are 
the  polymorphonuclear  leukocytes  of  the  blood,  and 
appear  as  rounded  cells  with  several  nuclei  or  one  very 
irregular  nucleus  (Fig.  11  and  Plate  II,  Fig.  2).     They 


6o  THE  SPUTUM 

are  frequently  filled  with  granules  of  coal-dust  and  are 
often  much  degenerated.  Such  coal-dust-laden  leuko- 
cytes are  especially  abundant  in  anthracosis,  where 
angular  black  particles,  both  intra-  and  extra-cellular, 
are  often  so  numerous  as  to  color  the  sputum  (Plate  II, 
Fig.  2,  B).  Occasionally  mononuclear  leukocytes  are 
present. 

Eosinophilic  cells  are  quite  constantly  found  in  large 
numbers  in  the  sputum  of  bronchial  asthma  near  the 


Fig.  15. — Sputum  from  a  case  of  asthma  showing'  Ijukocytes,  some  containiivg  eosino- 
philic granules;  free  eosinophilic  granules  and  micrococci;  stained  with  eosin  and  methy- 
lene-blue  ( X  350)  (Jakob). 

time  of  the  paroxysm,  and  constitute  one  of  the  most 
distinctive  features  of  the  sputum  of  this  disease.  They 
resemble  ordinary  pus-corpuscles,  except  that  their 
cytoplasm  is  filled  with  coarse  granules  having  a  marked 
affinity  for  eosin.  It  is  worthy  of  note  that  many  of 
them,  sometimes  the  majority,  are  mononuclear.  Large 
numbers  of  free  granules,  derived  from  disintegrated 
cells,  are  also  found  (Fig.  15). 


MICROSCOPIC  EXAMINATION  6l 

Ordinary  pus-cells  are  easily  recognized  in  sputum 
stained  by  any  of  the  methods  already  given.  For 
eosinophilic  cells,  some  method  which  includes  eosin  must 
be  used.  A  simple  method  is  to  stain  the  dried  and 
fixed  film  two  or  three  minutes  with  saturated  solution 
of  eosin,  and  then  one-half  to  one  minute  with  Loffler's 
methylene-blue ;  nuclei  and  bacteria  will  be  blue,  eosino- 
phiHc  granules  bright  red. 

(2)  Epithelial  cells  may  come  from  any  part  of  the 
respiratory  tract.  A  few  are  always  present,  since  des- 
quamation of  cells  goes  on  constantly.  Their  recogni- 
tion is  important  chiefly  as  an  aid  in  deciding  upon  the 
source  of  the  portion  of  the  sputum  in  which  they  are 
found.  In  suspected  lung  conditions  it  is  manifestly 
useless  to  study  material  from  the  nose  only,  yet  this 
is  not  infrequently  done.  They  have  little  diagnostic 
value,  although  a  considerable  excess  would  indicate  a 
pathologic  condition  at  the  site  of  their  origin.  Any 
of  the  stains  mentioned  above  will  show  them,  and  they 
can  usually  be  identified  in  unstained  sputum.  In 
general,  three  forms  are  found: 

(a)  Squamous  Cells. — Large,  flat,  polygonal  cells  with 
a  comparatively  small  nucleus  (Fig.  16,  i).  They  come 
from  the  upper  air-passages,  and  are  especially  numerous 
in  laryngitis  and  pharyngitis.  They  are  frequently 
studded  with  bacteria — most  commonly  diplococci. 

{h)  Cylindric  Cells  from  the  Nose,  Trachea,  and  Bronchi 
(Fig.  16,  /,  h). — These  are  not  usually  abundant,  and, 
as  a  rule,  they  are  not  identified  because  much  altered 
from  their  original  form,  being  usually  round  and  swollen. 
When  very  fresh,  they  may  retain  their  cylindric  form, 
sometimes  bearing  cilia  in  active  motion. 


62 


THE    SPUTUM 


(c)  Alveolar  Cells. — Rather  large,  round,  or  oval  cells 
with  one  or  two  round  nuclei  (Fig.  i6).  Their  source  is 
presumably  the  pulmonary  alveoli.  Like  the  leuko- 
cytes, they  frequently  contain  particles  of  carbon  (nor- 
mal lung  pigment).  In  chronic  heart  disease,  owing  to 
long-continued  passive  congestion,  they  may  be  filled 


Fig.  i6. — Different  morphologic  elements  of  the  sputum  (unstained):  a,  b,  c.  Pulmo- 
nary or  alveolar  epithelium — a,  with  normal  lung  pigment  (carbon);  6,  with  fat-droplets; 
c,  with  myelin  globules;  d,  pus -corpuscles;  e.  red  blood-corpuscles;  /,  cylindric  beaker- 
shaped  bronchial  epithelial  cells;  g,  free  myelin  globules;  h.  ciliated  epithelium  of  different 
kinds  from  the  nose,  altered  by  coryza;  i,  squamous  cells  from  the  pharynx  (after  Bizzo- 
zero). 


with  brown  granules  of  altered  blood-pigment,  and  are 
then  called  "  heart-failure  cells."  The  presence  of 
these  cells  in  considerable  numbers,  by  directing  one's 
attention  to  the  heart,  will  sometimes  clear  up  the 
etiology  of  a  chronic  bronchitis.  They  are  best  seen  in 
unstained   sputum,   appearing  as  grayish   or   colorless 


CHEMIC  EXAMINATION  63 

balls  filled  with  rounded  granules  of  brown  or  yellow 
pigment.     (Plate  II,  Fig.  i.) 

Alveolar  cells  commonly  contain  fat-droplets  and,  less 
frequently,  myelin  globules.  The  latter  are  colorless, 
rounded  bodies,  sometimes  resembUng  fat-droplets,  but 
often  showing  concentric  or  irregularly  spiral  markings 
(Fig.  16,  c,  g).  They  are  also  found  free  in  the  sputum. 
They  are  abundant  in  the  scanty  morning  sputum  of 
apparently  healthy  persons,  but  may  be  present  in  any 
mucoid  sputum. 

(3)  Red  blood-corpuscles  may  be  present  in  small 
numbers  in  almost  any  sputum.  When  fairly  constantly 
present  in  considerable  numbers,  they  are  suggestive  of 
phthisis.  The  corpuscles,  when  fresh,  are  shown  by  any 
of  the  staining  methods  which  include  eosin.  They  are 
commonly  so  much  degenerated  as  to  be  unrecognizable, 
and  often  only  altered  blood-pigment  is  left.  Ordinarily, 
blood  in  the  sputum  is  sufficiently  recognized  with  the 
naked  eye. 

III.  CHEMIC  EXAMINATION 

There  is  little  to  be  learned  from  a  chemic  examina- 
tion, and  it  is  rarely  undertaken.  Recently,  however, 
it  has  been  shown  that  the  presence  or  absence  of  albumin 
may  have  clinical  significance.  Albumin  is  constantly 
present  in  the  sputum  in  pneumonia,  pulmonary  edema, 
and  tuberculosis.  It  is  usually  absent  in  bronchitis. 
A  test  for  albumin  may,  therefore,  be  of  great  value  in 
distinguishing  between  bronchitis  and  tuberculosis,  a 
negative  result  practically  proving  the  absence  of  tuber- 
culosis. It  is  carried  out  as  follows:  The  sputum  is 
acidified  with  acetic  acid  to  precipitate  mucin  and  fil- 


64  THE   SPUTUM 

tered.  If  tenacious,  it  is  first  shaken  up  with  water. 
The  filtrate  is  then  tested  for  albumin,  as  described  in  the 
chapter  upon  the  Urine. 

IV.  THE  SPUTUM  IN  DISEASE 

Strictly  speaking,  any  appreciable  amount  of  sputum 
is  abnormal.  A  great  many  healthy  persons,  however, 
raise  a  small  quantity  each  morning,  owing  chiefly  to 
the  irritation  of  inhaled  dust  and  smoke.  Although 
not  normal,  this  can  hardly  be  spoken  of  as  pathologic. 
It  is  particularly  frequent  in  city  dwellers  and  in  those 
who  smoke  cigarettes  to  excess.  In  the  latter,  the 
amount  is  sometimes  so  great  as  to  arouse  suspicion  of 
tuberculosis.  Such  ''  normal  morning  sputum  "  gen- 
erally consists  of  small,  rather  dense,  mucoid  masses, 
translucent  white,  or,  when  due  to  inhaled  smoke,  gray 
in  color.  Microscopically,  there  are  a  few  pus-cor- 
puscles, and,  usually,  many  alveolar  cells,  both  of  which 
may  contain  carbon  particles.  The  alveolar  cells  com- 
monly show  myelin  degeneration,  and  free  myeHn  glob- 
ules may  be  present  in  large  numbers.  Saprophytic 
bacteria  may  be  present,  but  are  not  abundant. 

1.  Acute  Bronchitis. — There  is  at  first  a  small  amount 
of  tenacious,  almost  purely  mucoid  sputum,  frequently 
blood  streaked.  This  gradually  becomes  more  abun- 
dant, mucopurulent  in  character,  and  yellowish  or  gray 
in  color.  At  first  the  microscope  shows  a  few  leuko- 
cytes and  alveolar  and  bronchial  cells;  later,  the  leuko- 
cytes become  more  numerous.  Bacteria  are  not  usually 
abundant. 

2.  Chronic  Bronchitis.— The  sputum  is  usually  abun- 
dant, mucopurulent,  and  yellowish  or  yellowish-green  in 


THE   SPUTUM  IN  DISEASE  65 

color.  Nummular  masses — circular,  "  coin-like  "  discs 
which  sink  in  water — may  be  seen.  Microscopically, 
there  are  great  numbers  of  leukocytes,  often  much  degen- 
erated. Epithelium  is  not  abundant.  Bacteria  of 
various  kinds,  especially  staphylococci,  are  usually  nu- 
merous. 

In  fibrinous  bronchitis  there  are  found,  in  addition, 
fibrinous  casts,  usually  of  medium  size. 

In  the  chronic  bronchitis  accompanying  long-continued 
passive  congestion  of  the  lungs,  as  in  poorly  compensated 
heart  disease,  the  sputum  may  assume  a  rusty  brown 
color,  owing  to  presence  of  large  numbers  of  the  "  heart- 
failure  cells  "  previously  mentioned. 

3.  Bronchiectasis.— When  there  is  a  single  large 
cavity,  the  sputum  is  very  abundant  at  intervals, — 
sometimes  as  high  as  a  liter  in  twenty-four  hours, — and 
has  a  very  offensive  odor.  It  is  thinner  than  that  of 
chronic  bronchitis,  and  upon  standing  separates  into 
three  layers  of  pus,  mucus,  and  frothy  serum.  It 
contains  great  numbers  of  miscellaneous  bacteria. 

4.  Gangrene  of  the  Lung.— The  sputum  is  abun- 
dant, fluid,  very  offensive,  and  brownish  in  color.  It 
separates  into  three  layers  upon  standing^a  brown 
deposit,  a  clear  fluid,  and  a  frothy  layer.  Microscopic- 
ally, few  cells  of  any  kind  are  found.  Bacteria  are  ex- 
tremely numerous;  among  them  may  sometimes  be 
found  an  acid-fast  bacillus  probably  identical  with  the 
smegma  bacillus.  As  before  stated,  elastic  fibers  are 
usually  present  in  large  fragments. 

5.  Pulmonary  Edema.— Here  there  is  an  abundant, 
watery,  frothy  spfutum,  varying  from  faintly  yellow  or 
pink   to   dark  brown  in  color;  a  few  leukocytes  and 

5 


66  THE   SPUTUM 

epithelial  cells  and  varying  numbers  of  red  blood-cor- 
puscles are  found  with  the  microscope. 

6.  Bronchial  Asthma.— The  sputum  during  and  fol- 
lowing an  attack  is  scanty  and  very  tenacious.  Most 
characteristic  is  the  presence  of  Curschmann's  spirals, 
Charcot-Leyden  crystals,  and  eosinophilic  leukocytes. 

7.  Croupous  Pneumonia.— Characteristic  of  this  dis- 
ease is  a  scanty,  rusty  red,  very  tenacious  sputum,  con- 
taining red  corpuscles  or  altered  blood-pigment,  leuko- 
cytes, epithelial  cells,  usually  many  pneumococci,  and 
often  ver}'  small  fibrinous  casts.  This  sputum  is  seen 
during  the  stage  of  red  hepatization.  During  resolu- 
tion the  sputum  assumes  the  appearance  of  that  of  chronic 
bronchitis.  When  pneumonia  occurs  during  the  course 
of  a  chronic  bronchitis,  the  characteristic  rusty  red 
sputum  may  not  appear. 

8.  Pulmonary  Juberculosis.^The  sputum  is  varia- 
ble. In  the  earliest  stages  it  may  be  scanty  and  almost 
purely  mucoid,  with  an  occasional  yellow  flake,  or  there 
may  be  only  a  very  small  mucopurulent  mass.  When 
the  quantity  is  very  small,  there  may  be  no  cough,  the 
sputum  reaching  the  larynx  by  action  of  the  bronchial 
cilia.  This  is  not  well  enough  recognized  by  practi- 
tioners. A  careful  inspection  of  all  the  sputum  brought 
up  by  the  patient  on  several  successive  days,  and  a 
microscopic  examination  of  all  yellow  portions,  will  not 

"infrequently  establish  a  diagnosis  of  tuberculosis  when 
physical  signs  are  negative.  Tubercle  bacilli  will  some- 
times be  found  in  large  numbers  at  this  stage.  Blood- 
streaked  sputum  is  strongly  suggestive  of  tuberculosis, 
and  is  more  common  in  the  early  stages  than  later. 
The  sputum  of  more  advanced  cases  resembles  that  of 


THE   SPUTUM   IN   DISEASE  67 

chronic  bronchitis,  with  the  addition  of  tubercle  bacilli 
and  elastic  fibers.  Caseous  particles  containing  im- 
mense numbers  of  the  bacilli  are  common.  Far- 
advanced  cases  with  large  cavities  often  show  rather 
firm,  spheric  or  ovoid  masses  of  thick  pus  in  a  thin  fluid 
— the  so-called  "  globular  sputum."  These  globular 
masses  usually  contain  many  tubercle  bacilli.  Con- 
siderable hemorrhages  are  not  infrequent,  and  for  some 
time  thereafter  the  sputum  may  contain  clots  of  blood 
or  be  colored  brown. 


CHAPTER  II 

THE  URINE 

Preliminary  Considerations. — The  urine  is  an  aqueous 
solution  of  various  organic  and  inorganic  substances. 
It  is  probably  both  a  secretion  and  an  excretion.  Most 
of  the  substances  in  solution  are  either  waste-products 
from  the  body  metabolism  or  products  derived  directly 
from  the  foods  eaten.  Normally,  the  total  amount  of 
solid  constituents  carried  off  in  twenty-four  hours  is  about 
60  gm.,  of  which  the  organic  substances  make  up  about 
35  gm.  and  the  inorganic  about  25  gm. 

The  most  important  organic  constituents  are  urea, 
uric  acid,  and  ammonia.  Urea  constitutes  about  one- 
half  of  all  the  solids,  or  about  30  gm.  in  twenty-four 
hours. 

The  chief  inorganic  constituents  are  the  chlorids, 
phosphates,  and  sulphates.  The  chlorids,  practically 
all  in  the  form  of  sodium  chlorid ,  make  up  about  one-half 
of  the  inorganic  substances,  or  about  13  gm.,  in  twenty- 
four  hours. 

Certain  substances  appear  in  the  urine  only  in  patho- 
logic conditions.  The  most  important  of  these  are  pro- 
teins, sugars,  acetone,  and  related  substances,  bile,  hemo- 
globin, and  the  diazo  substances. 

In  addition  to  the  substances  in  solution  all  urines 
contain  various  microscopic  structures. 

While,  under  ordinary  conditions,  the  composition  of 

68 


THE  URINE  69 

urine  does  not  vary  much  from  day  to  day,  it  varies 
greatly  at  different  hours  of  the  same  day.  It  is  evident, 
therefore,  that  no  quantitative  test  can  be  of  value  unless  a 
sample  of  the  mixed  twenty-four-hour  urine  be  used.  The 
patient  should  be  instructed  to  void  all  the  urine  during 
the  twenty-four  hours  into  a  clean  vessel  kept  in  a  cool 
place,  to  mix  it  well,  to  measure  the  whole  quantity,  and 
to  bring  eight  or  more  ounces  for  examination,  A  pint 
fruit-jar  is  a  convenient  container.  When  it  is  desired 
to  make  only  qualitative  tests,  as  for  albumin  or  sugar,  a 
"  sample  "  voided  at  random  will  answer.  It  should  be 
remembered,  however,  that  urine  passed  about  three 
hours  after  a  meal  is  most  likely  to  contain  pathologic 
substances.  That  voided  first  in  the  morning  is  least 
likely  to  contain  them.  To  diagnose  cyclic  albuminuria 
samples  obtained  at  various  periods  during  the  twenty- 
four  hours  must  be  examined. 

The  urine  must  be  examined  while  fresh.  Decom- 
position sets  in  rapidly,  especially  in  warm  weather,  and 
greatly  interferes  with  all  the  examinations.  Decom- 
position may  be  delayed  by  adding  five  grains  of  boric 
acid  (as  much  of  the  powder  as  can  be  heaped  upon  a 
ten-cent  piece)  for  each  four  ounces  of  urine.  Formalin, 
in  proportion  of  one  drop  to  four  ounces,  is  also  an  effi- 
cient preservative,  but  if  larger  amounts  be  used,  it  may 
give  reactions  for  sugar  and  albumin,  and  is  likely  to 
cause  a  precipitate  which  greatly  interferes  with  the 
microscopic  examination. 

Normal  and  abnormal  pigments,  which  interfere  with 
certain  of  the  tests,  can  be  removed  by  filtering  the  urine 
through  animal  charcoal,  or  precipitating  with  a  solu- 
tion of  acetate  of  lead  and  filtering. 


70  THE   URINE 

Certain  cloudy  urines  cannot  be  clarified  by  ordinary 
filtration  through  paper,  particularly  when  the  cloudi- 
ness is  due  to  bacteria.  Such  urines  can  usually  be 
rendered  perfectly  clear  by  adding  a  small  amount  of 
purified  talc,  shaking  well,  and  filtering. 

A  suspected  fluid  can  be  identified  as  urine  by  detect- 
ing any  considerable  quantity  of  urea  in  it  (p.  93). 
Traces  of  urea  may,  however,  be  met  with  in  ovarian  cyst 
fluid,  while  urine  from  very  old  cases  of  hydronephrosis 
may  contain  little  or  none. 

The  frequency  of  micturition  is  often  suggestive  in 
diagnosis.  Whether  it  is  unduly  frequent  can  best  be 
ascertained  by  asking  the  patient  whether  he  has  to  get 
up  at  night  to  urinate.  Increased  frequency  may  be 
due  to  restlessness;  to  increased  quantity  of  urine;  to 
irritability  of  the  bladder,  usually  an  evidence  of  cys- 
titis; to  obstruction  ("  retention  with  overflow");  or  to 
paralysis  of  the  sphincter. 

Clinical  examination  of  the  urine  may  conveniently 
be  considered  under  four  heads:  I.  Physical  examina- 
tion. 11.  Chemic  examination.  III.  Microscopic  ex- 
amination.    IV.  The  urine  in  disease. 

I.  PHYSICAL  EXAMINATION 

1.  Quantity. — The  quantity  passed  in  twenty-four 
hours  varies  greatly  with  the  amount  of  liquids  ingested, 
perspiration,  etc.  The  normal  may  be  taken  as  1000  to 
1500  c.c,  or  40  to  50  ounces. 

The  quantity  is  increased  (polyuria)  during  absorption 
of  large  serous  efTusions  and  in  many  nervous  conditions. 
It  is  usually  much  increased  in  chronic  interstitial  nephri- 
tis, diabetes  insipidus,  and  diabetes  melHtus.     In  these 


PHYSICAL  EXAMINATION  7 1 

conditions  a  permanent  increase  in  amount  of  urine  is 
characteristic — a  fact  of  much  value  in  diagnosis.  In 
diabetes  mellitus  the  urine  may,  though  rarely,  reach  the 
enormous  amount  of  50  liters. 

The  quantity  is  decreased  (oliguria)  in  severe  diarrhea ; 
in  fevers;  in  all  conditions  which  interfere  with  circula- 
tion in  the  kidney,  as  poorly  compensated  heart  disease ; 
and  in  the  parenchymatous  forms  of  nephritis.  In 
uremia  the  urine  is  usually  very  greatly  decreased  and 
may  be  entirely  suppressed  (anuria). 

2.  Color. — This  varies  considerably  in  health,  and 
depends  largely  upon  the  quantity  of  urine  voided.  The 
usual  color  is  yellow  or  reddish-yellow,  due  to  the  pres- 
ence of  several  pigments,  chiefly  urochrome.  Acid  urine 
is  generally  darker  than  alkaline.  In  recording  the 
color  Vogel's  scale  (see  Frontispiece)  is  very  widely  used, 
the  urine  being  filtered  and  examined  by  transmitted 
light  in  a  glass  three  or  four  inches  in  diameter. 

The  color  is  sometimes  greatly  changed  by  abnormal 
pigments.  Blood-pigment  gives  a  red  or  brown,  smoky 
color.  Urine  containing  bile  is  yellowish  or  brown,  with 
a  yellow  foam  when  shaken.  It  may  assume  a  greenish 
hue  after  standing,  owing  to  oxidation  of  bilirubin  into 
biliverdin.  Ingestion  of  small  amounts  of  methylene- 
blue  gives  a  pale  green;  large  amounts  give  a  marked 
blue.  Santonin  produces  a  yellow;  rhubarb,  senna, 
cascara,  and  some  other  cathartics,  a  brown  color; 
these  change  to  red  upon  addition  of  an  alkali,  and  if 
the  urine  be  alkaline  when  voided,  may  cause  suspicion 
of  hematuria.  Thymol  gives  a  yellowish-green.  Fol- 
lowing poisoning  from  phenol  and  related  drugs  the  urine 
may  have  a  normal  color  when  voided,  but  becomes  olive- 


72  THE    URINE 

green  to  brownish-black  upon  standing.  In  susceptible 
individuals  therapeutic  doses  of  creosote,  or  absorption 
from  carbolized  dressings,  may  cause  this  change.  Urine 
which  contains  melanin,  as  sometimes  in  melanotic 
sarcoma,  and  very  rarely  in  wasting  diseases,  also  be- 
comes brown  or  black  upon  long  standing.  A  similar 
darkening  upon  exposure  to  the  air  occurs  in  alkapto- 
nuria (p.  126). 

A  pale  greenish  urine  with  high  specific  gravity 
strongly  suggests  diabetes  mellitus. 

3.  Transparency. — Freshly  passed  normal  urine  is 
clear.  Upon  standing,  a  faint  cloud  of  mucus,  leuko- 
cytes, and  epithelial  cells  settles  to  the  bottom — the  so- 
called  "nubecula."  Abnormal  cloudiness  is  usually  due 
to  presence  of  phosphates,  urates,  pus,  blood,  or  bacteria. 

Amorphous  pJiosphates  are  precipitated  in  neutral  or 
alkaline  urine.  They  form  a  white  cloud  and  sediment, 
which  disappear  upon  addition  of  an  acid. 

Amorphous  urates  are  precipitated  only  in  acid  urine. 
They  form  a  white  or  pink  cloud  and  sediment  ("  brick- 
dust  deposit  "),  which  disappear  upon  heating. 

Pus  resembles  amorphous  phosphates  to  the  naked  eye. 
Its  nature  is  easily  recognized  wdth  the  microscope,  oi" 
by  adding  a  strong  solution  of  caustic  soda  to  the  sedi- 
ment, which  is  thereby  transformed  into  a  gelatinous 
mass  (Donne's  test). 

Blood  gives  a  reddish  or  brown,  smoky  color,  and  may 
be  recognized  with  the  microscope  or  by  tests  for  hemo- 
globin. 

Bacteria,  when  present  in  great  numbers,  give  a  uni- 
form cloud,  which  cannot  be  removed  by  ordinary  filtra- 
tion.    They  are  detected  with  the  microscope. 


PHYSICAL  EXAMINATION  73 

The  cloudiness  of  decomposing  urine  is  due  mainly  to 
precipitation  of  phosphates  and  multiplication  of  bac- 
teria. 

4.  Odor. — The  characteristic  aromatic  odor  is  due  to 
volatile  acids,  and  is  most  marked  in  concentrated  urines. 
During  decomposition,  the  odor  becomes  ammoniacal. 
A  fruity  odor  is  sometimes  noted  in  diabetes,  due  prob- 
ably to  acetone.  Urine  which  contains  cystin  may  de- 
velop an  odor  of  sulphureted  hydrogen  during  decom- 
position. 

Various  articles  of  diet  and  drugs  impart  peculiar 
odors.  Notable  among  these  are  asparagus,  which  gives 
a  characteristic  oflfensive  odor,  and  turpentine,  which 
imparts  an  odor  somewhat  suggesting  that  of  violets. 

5.  Reaction. — Normally,  the  mixed  twenty-four-hour 
urine  is  slightly  acid  in  reaction.  The  acidity  was 
formerly  held  to  be  due  to  acid  phosphates,  but  Folin 
has  shown  that  the  acidity  of  a  clear  urine  is  ordinarily 
much  greater  than  the  acidity  of  all  the  phosphates,  the 
excess  being  due  to  free  organic  acids.  Individual 
samples  may  be  slightly  alkaline,  especially  after  a  full 
meal,  or  amphoteric.  The  reaction  is  determined  by 
means  of  htmus-paper. 

Acidity  is  increased  after  administration  of  certain 
drugs,  and  whenever  the  urine  is  concentrated  from  any 
cause,  as  in  fevers.  A  very  acid  urine  may  cause  fre- 
quent micturition  because  of  its  irritation.  This  is  often 
an  important  factor  in  the  troublesome  enuresis  of 
children. 

The  urine  always  becomes  alkahne  upon  long  standing, 
owing  to  decomposition  of  urea  with  formation  of  am- 
monia.     If  markedly  alkaline  when  voided,  it  usually 


74  THE    URINE 

indicates  such  "  ammoniacal  decomposition "  in  the 
bladder,  which  is  the  rule  in  chronic  cystitis,  especially 
that  due  to  paralysis  or  obstruction.  Alkalinity  due  to 
ammonia  (volatile  alkalinity)  can  be  distinguished  by 
the  fact  that  litmus  paper  turned  blue  by  the  urine  again 
becomes  red  upon  gentle  heating.  Fixed  alkalinity  is 
due  to  alkaline  salts,  and  is  often  observ^ed  during  fre- 
quent vomiting,  after  the  crisis  of  pneumonia,  in  various 
forms  of  anemia,  after  full  meals,  and  after  administra- 
tion of  certain  drugs,  especially  salts  of  vegetable  acids. 
Quantitative  estimation  of  the  acidity  of  urine  is  not 
of  much  clinical  value.  When,  however,  it  is  desired 
to  make  it,  the  method  of  Folin  will  be  found  satis- 
factory. In  every  case  the  sample  must  be  from  the 
mixed  twenty-four-hour  u^ine  and  as  fresh  as  possible. 

Folin's  Method. — Into  a  small  flask  measure  25  c.c.  of 
the  urine  and  add  i  or  2  drops  0.5  per  cent,  alcoholic  solu- 
tion of  phenolphthalein  and  15  or  20  gm.  of  neutral  potassium 
oxalate.  Shake  for  a  minute,  and  immediately  titrate  with 
decinormal  sodium  hydroxid,  shaking  meanwhile,  until  the 
first  permanent  pink  appears.  Read  off  from  the  buret  the 
amount  of  decinormal  sodium  hydroxid  solution  added,  and 
calculate  the  number  of  cubic  centimeters  which  would  be 
required  for  the  entire  twenty-four  hours'  urine.  Folin 
places  the  normal  acidity,  obtained  in  this  way,  at  617. 

6.  Specific  Gravity— The  normal  average  is  about 
1. 01 7  to  1.020.  Samples  of  urine  taken  at  random  may 
go  far  above  or  below  these  figures,  hence  a  sample  of  the 
mixed  twenty-four-hour  urine  should  always  be  used. 

Pathologically,  it  may  vary  from  i.ooi  to  1.060.  It  is 
low  in  chronic  interstitial  nephritis,  diabetes  insipidus, 


PHYSICAL  EXAMINATION 


75 


and  many  functional  nervous  disorders.  It  is  high  in 
fevers  and  in  parenchymatous  forms  of  nephritis.  In 
any  form  of  nephritis  a  sudden  fall  without  a  corre- 
sponding increase  in  quantity  of  urine  may  foretell  ap- 
proaching uremia.  It  is  highest  in  diabetes  melUtus.  A 
high  specific  gravity  when  the  urine  is  not  highly  colored 
should  lead  one  to  suspect  this  disease.  A  normal 
specific  gravity  does  not,  however,  exclude  it. 


l! 


I 


Fig.  17. — Squibb's  urinometer  with  thermometer  and  cylinder. 


The  specific  gravity  is  most  conveniently  estimated  by 
means  of  the  urinometer — Squibb's  is  preferable  (Fig. 
17).  It  is  standardized  for  a  temperature  of  77°  F.,  and 
the  urine  should  be  at  or  near  that  temperature.  Care 
should  be  taken  that  the  urinometer  does  not  touch  the 
side  of  the  tube,  and  that  air-bubbles  are  removed  from 
the  surface  of  the  urine.  With  most  instruments  the 
reading  is  taken  from  the  bottom  of  the  meniscus.  A 
long  scale  on  the  stem  is  desirable,  because  of  the  greater 
ease  of  accurate  reading. 


76 


THE   URINE 


One  frequently  wishes  to  ascertain  the  specific  gravity 
of  quantities  of  fluid  too  small  to  float  an  urinometer. 
A  simple  device  for  this  purpose,  which  requires  only 
about  3  c.c.  and  is  very  satisfactory  in  clinical  work  has 
been  designed  by  Saxe  (Fig.  i8).  The  urine  is  placed  in 
the  bulb  at  the  bottom,  the  instrument  is  floated  in  dis- 


Fig.  i8. — Saxe's  urinopyknometer  and  jar  for  same. 

tilled  water,  and  the  specific  gravity  is  read  off  from  the 
scale  upon  the  stem. 

7.  Total  Solids. — An  estimation  of  the  total  amount 
of  solids  which  pass  through  the  kidneys  in  twenty-four 
hours  is,  in  practice,  one  of  the  most  useful  of  urinary 
examinations.  The  normal  for  a  man  of  150  pounds  is 
about  60  gm.,  or  950  gr.     The  principal  factors  which 


PHYSICAL  EXAMINATION  77 

influence  this  amount  are  body  weight  (except  with 
excessive  fat),  diet,  exercise,  and  age,  and  these  should 
be  considered  in  making  an  estimation.  After  about 
the  f orty-fif th  year  it  becomes  gradually  less ;  after 
seventy-five  years  it  is  about  one-half  the  amount  given. 

In  disease,  the  amount  of  solids  depends  mainly  upon 
the  activity  of  metabolism  and  the  ability  of  the  kidneys 
to  excrete.  An  estimation  of  the  solids,  therefore, 
furnishes  an  important  clue  to  the  functional  efficiency 
of  the  kidneys.  The  kidneys  bear  much  the  same 
relation  to  the  organism  as  does  the  heart:  they  cause 
no  direct  harm  so  long  as  they  are  capable  of  perform- 
ing the  work  required  of  them.  When,  however,  through 
either  organic  disease  or  functional  inactivity,  they  fail 
to  carry  off  their  proportion  of  the  waste-products  of  the 
body,  some  of  these  products  must  either  be  eliminated 
through  other  organs,  where  they  cause  irritation  and 
disease,  or  be  retained  within  the  body,  where  they  act 
as  poisons.  The  great  importance  of  these  poisons  in 
production  of  distressing  symptoms  and  even  organic 
disease  is  not  well  enough  recognized  by  most  practi- 
tioners. Disappearance  of  unpleasant  and  perplexing 
symptoms  as  the  urinary  solids  rise  to  the  normal  under 
proper  treatment  is  often  most  surprising. 

When,  other  factors  remaining  unchanged,  the 
amount  of  solids  eliminated  is  considerably  above  the 
normal,  increased  destructive  metabolism  may  be 
inferred. 

The  total  solids  can  be  estimated  roughly,  but  ac- 
curately enough  for  most  clinical  purposes,  by  multi- 
plying the  last  two  figures  of  the  specific  gravity  of  the 
mixed  twenty-four-hour  urine  by  the  number  of  ounces 


78  THE    URINE 

voided  and  to  the  product  adding  one-tenth  of  itself. 
This  gives  the  amount  in  grains.  If,  for  example,  the 
twenty-four-hour  quantity  is  3  pints  or  48  ounces,  and 
the  specific  gravity  is  1.018,  the  total  solids  would  ap- 
proximate 950  gr.,  as  follows: 

48  X  18  =  864;  864  +  86.4  -  950.4 

This  method  is  especially  convenient  for  the  practi- 
tioner, because  patients  nearly  always  report  the  amount 
of  urine  in  pints  and  ounces,  and  it  avoids  the  necessity 
of  converting  into  the  metric  system.  Haser's  method  is 
more  widely  used  but  is  less  convenient.  The  last  two 
figures  of  the  specific  gravity  are  multiplied  by  2.33. 
The  product  is  then  multiplied  by  the  number  of  cubic 
centimeters  voided  in  twenty-four  hours  and  divided 
by  1000.     This  gives  the  total  solids  in  grams. 

8.  Functional  Tests.— Within  the  past  few  years 
much  thought  has  been  devoted  to  methods  of  more 
accurately  ascertaining  the  functional  efficiency  of  the 
kidneys,  especially  of  one  kidney  when  removal  of  the 
other  is  under  consideration.  The  most  promising  of 
the  methods  which  have  been  devised  are  cryoscopy, 
electric  conductivity,  the  methylene-blue  test,  and  the 
phloridzin  test.  It  is  doubtful  whether,  except  in 
experienced  hands,  these  yield  any  more  information 
than  can  be  had  from  an  intelligent  consideration  of  the 
specific  gravity  and  the  twenty-four-hour  quantity,  to- 
gether wath  a  microscopic  examination.  They  are  most 
useful  when  the  urines  obtained  from  separate  kidneys 
by  segregation  or  ureteral  catheterization  are  compared. 
The  reader  is  referred  to  larger  works  upon  urinalysis 
for  details. 


PHYSICAL  EXAMINATION  79 

Cryoscopy,  determination  of  the  freezing-point,  de- 
pends upon  the  principle  that  the  freezing-point  of  a 
fluid  is  depressed  in  proportion  to  the  number  of  mole- 
cules, organic  and  inorganic,  in  solution.  To  have  any 
value,  the  freezing-point  of  the  urine  must  be  compared 
with  that  of  the  blood,  since  it  is  not  so  much  the  num- 
ber of  molecules  contained  in  the  urine  as  the  number 
which  the  kidney  has  failed  to  carry  off  and  has  left  in 
the  blood,  that  indicates  its  insufficiency. 

Electric  conductivity  refers  to  the  power  of  the  urine 
to  carry  an  electric  current.  It  is  increased  in  pro- 
portion to  the  nimiber  of  inorganic  molecules  in  solution. 

In  the  methylene-blue  test  of  Achard  and  Castaigne 
a  solution  of  methylene-blue  is  injected  intramuscularly, 
and  the  time  of  its  appearance  in  the  urine  is  noted. 
Normally,  it  appears  in  about  thirty  minutes.  When 
delayed,  renal  "  permeability  "  is  supposed  to  be  inter- 
fered with.  Since  methylene-blue  is  sometimes  ex- 
creted as  a  colorless  derivative,  indigo-carmin  has  been 
proposed  as  a  substitute.  In  the  absence  of  renal  in- 
sufficiency this  always  gives  a  blue  color,  which  begins 
to  appear  in  about  five  minutes. 

The  phloridzin  test  consists  in  the  hypodermic  injec- 
tion of  a  small  quantity  of  phloridzin.  This  substance 
is  transformed  into  glucose  by  the  kidneys  of  healthy 
persons.  In  disease,  this  change  is  more  or  less  inter- 
fered with,  and  the  amount  of  glucose  recoverable  from 
the  urine  is  taken  as  an  index  of  the  secretory  power  of 
the  kidneys. 

In  applying  these  tests  for  "  permeability,"  "  secre- 
tory ability,"  etc.,  one  must  rertiember  that  the  condi- 
tions are  abnormal,  and  that  there  is  no  e\ddence  that 


8o  THE    LTIIXE 

the  kidneys  will  behave  with  the  products  of  metabolism 
as  they  do  with  the  substances  selected  for  the  tests,  and 
also  that  the  tests  throw  unusual  work  upon  the  kidneys, 
which  in  some  cases  may  be  harmful. 

II.  CHEMIC  EXAMINATION 

A.    Normal  Constituents 

Of  the  large  number  of  organic  and  inorganic  sub- 
stances normally  present  in  the  urine,  only  a  few  demand 
any  consideration  from  the  clinician.  The  following 
table,  therefore,  outlines  the  average  composition  from 
the  clinical,  rather  than  from  the  chemical,  standpoint. 
Only  the  twenty-four-hour  quantities  are  given,  since 
they  alone  furnish  an  accurate  basis  for  comparison. 
The  student  tannot  too  soon  learn  that  percentages  mean 
little  or  nothing,  excepting  as  they  furnish  a  means  of 
calculating  the  twenty-four-hour  elimination. 

COMPOSITION  OF  NORMAL  URINE 

Grams  in  twenty-  Approximate 

four  hours.  average. 

Total  substances  in  solution 55-70  60 

Inorganic  substances 20-30  25 

Chlorids  (chiefly  sodium  chlorid) 10-15  12.5 

Phosphates  (estimated  as  phosphoric  acid), 

total 2.5-3.5  3 

Earthy  3  of  total i 

Alkaline  §  of  total 2 

Sulphates  (estimated  as  sulphuric  acid),  total  1.5-3.0  2.5 

Mineral  j%  of  total 2.25 

Conjugate  yV  of  total 0.25 

Includes  indiran Trace 

Ammonia 0.5-1 .0  0.7 

Organic  substances 30-40  35 

Urea 20-35  3° 

Uric  acid 0.4-1.0  0.7 


CHEMIC  EXAMINATION 


8l 


Fig.  iQ. — Graphic  expression  of  quantities  in  the  urine.    Solid  line,  normal  urine;  dotted 
line,  an  example  of  pathologic  urine  in  a  case  of  cancerous  cachexia  (Saxe). 


82  THE   URINE 

Although  the  conjugate  sulphates  are  organic  com- 
pounds, they  are,  for  the  sake  of  convenience,  included 
with  the  inorganic  sulphates  in  the  table  on  p.  80. 

Among  constituents  which  are  of  little  clinical  im- 
portance, or  are  present  only  in  traces,  are: 

Inorganic:  Iron,  carbonates,  nitrates,  silicates,  and 
fiuorids. 

Organic:  Creatinin,  hippuric  acid,  purin  bases,  oxalic 
acid,  benzoic  acid,  volatile  fatty  acids,  pigments,  and 
acetone. 

Variations  in  body  weight,  diet,  and  exercise  cause 
marked  fluctuations  in  the  total  solids  and  in  individual 
substances. 

1.  Chlorids. — These  are  derived  from  the  food,  and 
are  mainly  in  the  form  of  sodium  chlorid.  The  amount 
excreted  normally  is  10  to  15  gm.  in  twenty-four  hours. 
It  is  much  affected  by  the  diet,  and  is  reduced  to  a 
minimum  in  starvation. 

Excretion  of  chlorids  is  diminished  in  nephritis  and 
in  fevers,  especially  in  pneumonia  and  inflammations 
leading  to  the  formation  of  large  exudates.  In  nephritis 
the  kidneys  are  less  permeable  to  the  chlorids,  and  it  is 
possible  that  the  edema  is  due  largely  to  an  effort  of 
the  body  to  dilute  the  chlorids  which  have  been  retained. 
Certainly  an  excess  of  chlorids  in  the  food  will  increase 
both  the  albuminuria  and  the  edema  of  nephritis.  In 
fevers  the  diminution  is  due  largely  to  decrease  of  food, 
though  probably  in  some  measure  to  impaired  renal 
function.  In  pneumonia  chlorids  are  constantly  very 
low,  and  in  some  cases  are  absent  entirely.  Following 
the  crisis  they  are  increased.  In  inflammations  leading 
to   formation   of   large   exudates — e.   g.,   pleurisy   with 


CHEMIC  EXAMINATION 


83 


effusion — chlorids  are  diminished,  because  a  consider- 
able amount  becomes  "  locked  up  "  in  the  exudate. 
During  absorption  chlorids  are  liberated  and  appear 
in  the  urine  in  excessive  amounts. 

Diminution  of  chlorids  is  also  observed  in  severe 
diarrhea,  anemic  conditions,  and  carcinoma  of  the 
stomach. 


Fig.  20.— The  Purdy  electric  centrifuge  with  four  arras. 

Quantitative  Estimation. — The  best  method  for  clinical 
purposes  is  the  centrifugal  method. 

Purdy's  Centrifugal  Methods. — As  shown  by  the  late 
Dr.  Purdy,  the  centrifuge  offers  an  important  means 
of  making  quantitative  estimations  of  a  number  of  sub- 
stances in  the  urine.  Results  are  easily  and  quickly 
obtained,  and  are  probably  accurate  enough  for  all  clini- 
cal purposes. 

In  general,  the  methods  consist  in  precipitating  the 
substance  to  be  estimated  in  a  graduated  centrifuge  tube. 


84 


THE    URINE 


and  applying  a  definite  amount  of  centrifugal  force  for  a 
definite  length  of  time,  after  which  the  percentage  of 
precipitate  is  read  off  upon  the  side  of  the  tube.  Al- 
bumin, if  present,  must  be  previously  removed  by  boil- 
ing and  filtering.  Results  are  in  terms  of  bulk  of  pre- 
cipitate, which  must  not  be  confused  with  percentage 
by  weight.  The  weight  percentage  can  be  found  by 
referring  to  Purdy's  tables,  given  later.     In  this,  as  in 


Fis-  21. — Water-motor  centrifuge. 


all  quantitative  urine  work,  percentages  mean  little  in 
themselves;  the  actual  amount  eliminated  in  twenty- 
four  hours  should  always  be  calculated. 

The  centrifuge  should  have  an  arm  with  radius  of  6f 
inches  when  in  motion,  and  should  be  capable  of  main- 
taining a  speed  of  1500  revolutions  a  minute.  The 
electric  centrifuge  is  to  be  recommended,  although 
good  work  can  be  done  with  a  water-power  centrifuge, 
or,  after  a  little  practice,  with  the  hand  centrifuge.     A 


CHEMIC  EXAMINATION 


85 


speed  indicator  is  desirable  with  electric  and  water- 
motor  machines,  although  one  can  learn  to  estimate  the 
speed  by  the  musical  note. 


c.c\ 


Fig.  22. — Purdy's  tubes  for  the  centrifuge:  a,  Percentage  tube;  6,  sediment  tube. 


Estimation  of  Chlorids. — Fill  the  graduated  tube  to  the 
10  CO.  mark  with  urine;  add  15  drops  strong  nitric  acid  and 
then  silver  nitrate  solution  (dram  to  the  ounce)  to  the  15  c.c, 
mark.  Mix  by  inverting  several  times.  Let  stand  a  few 
minutes  for  a  precipitate  to  form,  and  then  revolve  in  the 
centrifuge  for  three  minutes  at  1200  revolutions  a  minute. 
Each  one-tenth  cubic  centimeter  of  precipitate  equals  i  per 
cent,  by  bulk.  The  normal  is  about  10  per  cent.  This  may 
be  converted  into  terms  of  chlorin  or  sodium  chlorid  by  means 
of  the  table  upon  page  86.     Roughly  speaking,  the  percent- 


86 


THE   URINE 


age  of  chlorin  by  weight  is  about  one-twelfth  the  bulk- 
percentage. 

TABLE  FOR  THE  ESTIMATION  OF   CHLORIDS  AFTER 
CENTRIFUGATION 

Showing  the  bulk-percentage  of  silver  chlorid  {AgCl)  and  the  correspond- 
ing gravimetric  percentages  and  grains  per  fluidounce  of  sodium  chlorid 
{NaCl)  and  chlorin  {CI). — (Ptirdy.) 


d 

C3 

u 

a 

d 

u 

0 

y 

u 

0 

2 

C 

"A 

N 

1-1 

V 

s 

a 

SO 

.',  0 

V 

be 

s 

K 

0 

g 

Ji   0 

^ 
b 

0 

iZ 

0 

3 

1 

c 

1^ 

0 

i 

0.03 

0.15 

0.02 

0.1    1 

8 

1.04 

4.98 

0.63 

3.02 

i 

0.07 

0.31 

0.04 

0.19 

82 

I.I 

5-29 

0.67 

3.22 

1 

O.I 

0.47 

0.06 

0.28 

9, 

1. 17 

5-6 

0.71 

3-4 

I 

0.13 

0.62 

0.08 

0.38 

9^ 

1.23 

5-91 

0-75 

3-6 

i\ 

0.16 

0.78 

O.I 

0.48 

10 

1-3 

6.22 

0.79 

3-79 

li 

0.19 

0-93 

0.12 

0-57 

10^ 

1.36 

6.53 

0.83 

3-97 

i| 

0.23 

1.09 

0.14 

0.67 

II 

1-43 

6.84 

0.87 

4.16 

2 

0.26 

1.24 

0.16 

0.76 

iij 

1.49 

7.2 

0.91 

4-35 

2^ 

0.29 

1.41 

0.18 

0.85 

12 

1.56 

7.46 

0-95 

4-54 

2j 

0.32 

1.56 

0.2 

0.96 

12J 

1.62 

7.78 

0.99 

4-73 

2| 

0.36 

1.71 

0.22 

1.04 

13, 

1.69 

8.09 

1.02 

4.92 

3 

0-39 

1.87 

0.24 

1-13 

13J 

1-75 

8.4 

1.06 

5-II 

3} 

0.42 

2.02 

0.26 

1.23 

14 

1.82 

8.71 

I.I 

529 

4 

0.4S 

2.18 

0.28 

1.32 

14J 

1.88 

9.02 

1. 14 

5-49 

3l 

0.49 

2-35 

0-3 

1.42 

15^ 

1.94 

9-33 

1. 18 

5-67 

4 

0.52 

2.49 

0.32 

1-51 

15^ 

2.01 

965 

1.22 

5-86 

4} 

0-55 

2.64 

0-34 

1.61 

16 

2.07 

9.94 

1.26 

6.06 

4* 

0.58 

2.8 

0-3S 

1-7 

16J 

2.14 

10.27 

1-3 

6.24 

4l 

0.62 

2.96 

0-37 

1.8 

'7, 

2.2 

10.151 

1-34 

6.43 

5, 

0.65 

3-II 

0-39 

1.89 

17J 

2.27 

10.87 

1.38 

6.62 

5h 

0.71 

3-42 

0.43 

2.09 

18 

2-33 

II. 2 

1.42 

6.81 

6 

0.78 

3-73 

0.47 

2.27 

18J 

2.4 

11.51 

1.46 

7.0 

6J 

0.84 

4-05 

0.51 

2.46 

19 

2.46 

11.82 

i-S 

7.19 

7, 

0.91 

4-35 

0-S5 

2.62 

19^ 

2-53 

12.13 

1-54 

7-38 

7i 

0.97 

4.67 

0-59 

2.84 

20 

2-59 

12.44 

1.58 

7-56 

Bulk-percentage  to  be  read  on  the  side  of  the  tube. 


2.  Phosphates. — Phosphates  are  derived  largely  from 
the  food,  only  a  small  proportion  resulting  from  metab- 


CHEMIC  EXAMINATION  87 

olism.  The  normal  daily  output  of  phosphoric  acid  is 
about  2.5  to  3.5  gm. 

The  urinary  phosphates  are  of  two  kinds:  alkaline, 
which  make  up  two-thirds  of  the  whole,  and  include  the 
phosphates  of  sodium  and  potassium;  and  earthy,  which 
constitute  one-third,  and  include  the  phosphates  of  cal- 
cium and  magnesium.  Earthy  phosphates  are  fre- 
quently thrown  out  of  solution  in  neutral  and  alkaline 
urines,  and  as  "  amorphous  phosphates  "  form  a  very 
common  sediment.  This  sediment  seldom  indicates  an 
excessive  excretion  of  phosphoric  acid.  It  is  usually 
merely  an  evidence  of  diminished  acidity  of  the  urine,  or 
of  an  increase  in  the  proportion  of  phosphoric  acid  elimi- 
nated as  earthy  phosphates.  This  form  of  "  phospha- 
turia  "  is  most  frequent  in  neurasthenia  and  hysteria. 
When  the  urine  undergoes  ammoniacal  decomposition, 
some  of  the  ammonia  set  free  combines  with  magnesium 
phosphate  to  form  ammoniomagnesium  phosphate 
("  triple  phosphate  "),  which  is  deposited  in  typical 
crystalline  form  (p.  148). 

Excretion  of  phosphates  is  increased  by  a  rich  diet; 
in  active  metabolism ;  in  certain  nervous  and  mental  dis- 
orders; in  leukemia;  and  in  phosphatic  diabetes,  an  ob- 
scure disturbance  of  metabolism  (not  related  to  diabetes 
mellitus)  which  is  associated  with  an  increase  in  the  out- 
put of  phosphates  up  to  10  gm.  or  more  in  twenty-four 
hours.  Phosphates  are  decreased  in  chronic  diseases 
with  lowered  metabolism;  in  hepatic  cirrhosis  and  acute 
yellow  atrophy;  in  pregnancy,  owing  to  developing 
fetal  bones;  and  in  nephritis,  owing  to  kidney  imper- 
meability. 

Quantitative   estimation    does    not    furnish    much    of 


88 


THE    URINE 


definite  clinical  value.     The  centrifugal  method  is  the 
most  convenient. 

T.\BLE  FOR  THE  ESTIMATION  OF  PHOSPHATES  AFTER 
CEXTRIFUGATION 

Showing  hulk-percentages  of  uranyi  phosphate  {H\UO^PO^  and  the  cor- 
responding gravimetric  percentages  and  grains  per  ounce  oj  phosphoric 
acid  {P.p^).—{Purdy.) 


Bulk-per- 

rentage  of 

H(U0„)P04. 

Percentage 
P.O5. 

Or.  Per  Oz. 
P2O5. 

Bulk-per- 
centage of 
H(U0.^)P04. 

1 1 

Percentage 

Gr.  Per  Oz. 
P-iO,. 

b 

0.02 

O.I 

0.14 

0.67 

I 

0.04 

0.19 

12 

o.is 

0.72 

ij 

0.045 

0.22 

13 

0.16 

0.77 

2 

0.05 

0.24 

14 

0.17 

0.82 

2 1 

0-055 

0.26 

IS 

0.18 

0.86 

3, 

0.06 

0.29 

16 

0.19 

0.91 

3i 

0.065 

0.31 

17 

0.2 

0.96 

4 

0.07 

0.34 

18 

0.21 

I. 

4i 

0.075 

0.36 

19 

0.22 

1.06 

5 

0.08 

0.38 

20 

0.23 

I.I 

6 

0.09 

0.43 

21 

0.24 

115 

/ 

O.I 

0.48 

22 

0.25 

1.2 

8 

O.II 

0-53 

^3 

0.26 

1-25 

9 

0.12 

0-58 

24 

0.27 

1-3 

lO 

0.13 

0.62      1 

25 

0.28 

1-35 

Bulk-percentage  to  be  read  from  graduation  on  the  side  of  the  tube. 

Purdy's  Centrifugal  Method. — Take  10  c.c.  urine  in  the 
graduated  tube,  add  2  c.c.  of  50  per  cent,  acetic  acid,  and  3  c.c. 
of  5  per  cent,  uranium  nitrate  solution.  Mix;  let  stand  a  few 
minutes,  and  revolve  for  three  minutes  at  1200  revolutions. 
The  bulk  of  precipitate  is  normally  about  8  per  cent.  The 
percentage  of  phosphoric  acid  by  weight  is,  roughly,  one- 
eighty-fifth  of  the  bulk-percentage. 

3.  Sulphates. — The  urinary  sulphates  are  derived 
partly  from  the  food,  especially  meats,  and  partly  from 
body  metabolism.  The  normal  output  of  sulphuric  acid 
is  about  1.5  to  3  gm.  daily.     It  is  increased  in  condi- 


CHEMIC  EXAMINATION 


89 


tions  associated  with  active  metabolism,  and  in  general 
may  be  taken  as  a  rough  index  of  protein  metabolism. 

Quantitative  estimation  of  the  total  sulphates  yields 
little  of  clinical  value. 


Purdy's  Centrifugal  Method. — Take  10  c.c.  urine  in  the 
graduated  tube  and  add  barium  chlorid  solution  to  the 
15  c.c.  mark.  This  consists  of  barium  chlorid,  4  parts; 
strong  hydrochloric  acid,  i  part;  and  distilled  water,  16 
parts.  Mix;  let  stand  a  few  minutes,  and  revolve  for  three 
minutes  at  1200  revolutions  a  minute.  The  normal  bulk 
of  precipitate  is  about  0.8  per  cent.  The  percentage  by 
weight  of  sulphuric  acid  is  about  one-fourth  of  the  bulk- 
percentage. 

TABLE  FOR  THE  ESTIMATION  OF  SULPHATES  AFTER 
CENTRIFUGATION 
Showing  the  hulk-percentages  of  barium  sulphate  (^BaSO^  and  the  cor- 
responding gravimetric  percentages  and  grains  per  fiuidounce  of  sul- 
phuric acid  (SO3). — (Purdy.) 


Bulk-per- 
centage of 
BaSO*. 

Percentage 
SO3. 

Gr.  Per  Oz. 
SO3. 

Bulk-per- 
centage of 
BaS04. 

Percentage 
SO3. 

Gr.  Per  Oz. 
SO3. 

■ 

0.04 

0.19 

2i 

0-55 

2.64 

:  ■ 

0.07 

0.34 

2i 

0.61 

2-93 

■ 

O.I 

0.48 

2| 

0.67 

3.22 

:  ■ 

0.13 

0.62 

3, 

0-73 

3-5 

0.16 

0.77 

3* 

0.79 

3-79 

0.19 

0.91 

4 

0.85 

4.08 

0.22 

1.06 

3l 

0.91 

4-37 

I 

0.25 

I.I 

4 

0.97 

4.66 

li 

0.31 

1.49 

4i 

1.03 

4.94 

^ 

0-37 

1.78 

4i 

1.09 

5-23 

l| 

0-43 

2.06 

4l 

i-iS 

5-52 

2 

0.49 

2-35 

5 

1. 21 

5-8i 

Bulk-percentage  to  be  read  from  graduation  on  the  side  of  the  tube. 

About  nine-tenths  of  the  sulphuric  acid  is  in  com- 
bination with  various  mineral  substances,  chiefly  sodium, 


90  THE   URINE 

potassium,  calcium,  and  magnesium  (mineral  or  pre- 
formed sulphates).  One-tenth  is  in  combination  with 
certain  aromatic  substances,  which  are  mostly  products 
of  albuminous  putrefaction  in  the  intestine,  but  are  de- 
rived in  part  from  destructive  metabolism  (conjugate 
or  ethereal  sulphates) .  Among  these  aromatic  substances 
are  indol,  phenol,  and  skatol.  By  far  the  most  impor- 
tant of  the  conjugate  sulphates  and  representative  of  the 
group  is  potassium  indoxyl  sulphate. 

Potassium  indoxyl  sulphate,  or  indican,  is  derived 
from  indol.  Indol  is  absorbed  and  oxidized  into  in- 
doxyl, which  combines  with  potassium  and  sulphuric 
acid  and  is  thus  excreted.  Under  normal  conditions 
the  amount  in  the  urine  is  small.  It  is  increased  by  a 
meat  diet. 

Unlike  the  other  ethereal  sulphates,  which  are  de- 
rived in  part  from  metabolism,  indican  originates  prac- 
tically wholly  from  putrefactive  processes.  It  alone, 
therefore,  and  not  the  total  ethereal  sulphates,  can  be 
taken  as  an  index  of  such  putrefaction.  A  pathologic 
increase  is  called  indicanuria.     It  is  noted  in: 

(a)  Diseases  of  the  Small  Intestine. — This  is  by  far 
the  most  common  source.  Intestinal  obstruction  gives 
the  largest  amounts  of  indican.  It  is  also  much  in- 
creased in  intestinal  indigestion — so-called  "  bilious- 
ness ";  in  inflammations,  especially  in  cholera  and  ty- 
phoid fever;  and  in  paralysis  of  peristalsis,  such  as 
occurs  in  peritonitis.  Simple  constipation  and  diseases 
of  the  large  intestine  alone  do  not  so  frequently  cause 
indicanuria. 

(b)  Diseases  of  the  stomach  associated  with  deficient 
hydrochloric  acid,  as  chronic  gastritis  and  gastric  cancer. 


CHEMIC  EXAMINATION  9 1 

Diminished  hydrochloric  acid  favors  intestinal  putre- 
faction. 

(c)  Diminished  Flow  of  Bile. — Since  the  bile  serves 
both  as  a  stimulant  to  peristalsis  and  an  intestinal  anti- 
septic, a  diminished  flow  from  any  cause  favors  occur- 
rence of  indicanuria. 

{d)  Decomposition  of  exudates  anywhere  in  the  body, 
as  in  empyema,  bronchiectasis,  and  large  tuberculous 
cavities. 

Detection  of  indican  depends  upon  its  decomposition 
and  oxidation  of  the  indoxyl  set  free  into  indigo-blue. 
This  change  sometimes  takes  place  spontaneously  in 
decomposing  urine,  causing  a  dirty  blue  color.  Crystals 
of  indigo  (Fig.  36)  may  be  found  both  in  the  sediment 
and  the  scum. 

Obermayer's  Method. — In  a  test-tube  take  equal  parts  of 
the  urine  and  Obermayer's  reagent  and  add  a  small  quantity 
of  chloroform.  Mix  by  inverting  a  few  times;  avoid  shak- 
ing violently.  If  indican  be  present  in  excess,  the  chloroform, 
which  sinks  to  the  bottom,  will  assume  an  indigo-blue  color. 
It  will  take  up  the  indigo  more  quickly  if  the  urine  be  warm. 
The  depth  of  color  indicates  the  comparative  amount  of 
indican  if  the  same  proportions  of  urine  and  reagents  are 
always  used,  but  one  should  bear  in  mind  the  total  amount 
of  urine  voided.  The  indican  in  normal  urine  may  give 
a  faint  blue  by  this  method.  Urine  of  patients  taking  iodids 
gives  a  reddish-violet  color,  which  disappears  upon  addition 
of  a  few  drops  of  strong  sodium  hyposulphite  solution  and 
shaking.  Bile-pigments,  which  interfere  with  the  test,  must 
be  removed  (p.  69), 

Obermayer^s  reagent  consists  of  strong  hydrochloric  acid 
(sp.  gr.,  1. 19),  1000  parts,  and  ferric  chlorid,  2  parts.  This 
makes  a  yellow,  fuming  liquid  which  keeps  well. 


92  THE   URINE 

4.  Urea. — From  the  standpoint  of  physiolog}-^  urea  is 
the  most  important  constituent  of  the  urine.  It  is  the 
principal  waste-product  of  metaboHsm,  and  constitutes 
about  one-half  of  all  the  solids  excreted — about  20  to 
35  gm.  in  twenty-four  hours.  It  represents  85  to  90 
per  cent,  of  the  total  nitrogen  of  the  urine,  and  its  quan- 
titative estimation  is  a  simple,  though  not  very  accurate, 
method  of  ascertaining  the  state  of  nitrogenous  excretion. 

This  is  true,  however,  only  in  normal  individuals 
upon  average  mixed  diet.  Under  pathologic  conditions, 
the  proportion  of  nitrogen  distributed  among  the  various 
nitrogen-containing  substances  undergoes  great  varia- 
tion. The  only  accurate  index  of  protein  metabolism 
is,  therefore,  the  total  output  of  nitrogen,  which  can  be 
estimated  by  the  Kjeldahl  method.  The  whole  subject 
of  "  nitrogen  partition  "  and  "  nitrogen  equilibrium  " 
(relation  of  excretion  to  intake)  is  an  important  one,  but 
is  out  of  the  province  of  this  book,  since  as  yet  it  con- 
cerns the  physiologic  chemist  more  than  the  clinician. 

It  may  be  helpful  to  state  here,  however,  that  upon  a  mixed 
diet  the  nitrogen  of  the  urine  is  distributed  about  as  follows: 
urea  nitrogen,  86.9  per  cent.;  ammonia  nitrogen,  4.4  per 
cent.;  creatinin  nitrogen,  3.6  per  cent.;  uric  acid  nitrogen, 
0.75  per  cent.;  "undetermined  nitrogen,"  chiefly  in  amino 
acids,  4.3  per  cent. 

Normally,  the  amount  is  greatly  influenced  by  ex- 
ercise and  diet.  It  is  increased  by  copious  drinking 
of  water  and  administration  of  ammonium  salts  of 
organic  acids. 

Pathologically,  urea  is  increased  in  fevers,  in  diabetes, 
and  especially  during  resolution  of  pneumonia  and  ab- 


CHEMIC  EXAMINATION  93 

sorption  of  large  exudates.  As  above  indicated,  when 
other  factors  are  equal,  the  amount  of  urea  indicates 
the  activity  of  metabolism.  In  deciding  whether  in  a 
given  case  an  increase  of  urea  is  due  to  increased  metab- 
ohsm  the  relation  between  the  amounts  of  urea  and  of 
the  chlorids  is  a  helpful  consideration.  The  amount 
of  urea  is  normally  about  twice  that  of  the  chlorids.  If 
the  proportion  is  much  increased  above  this,  increased 
tissue  destruction  may  be  inferred,  since  other  condi- 
tions which  increase  urea  also  increase  chlorids. 

Urea  is  decreased  in  diseases  of  the  liver  with  destruc- 
tion of  liver  substance,  such  as  cirrhosis,  carcinoma, 
and  acute  yellow  atrophy.  It  may  or  may  not  be 
decreased  in  nephritis.  In  the  early  stages  of  chronic 
nephritis,  when  diagnosis  is  difficult,  it  is  usually  normal. 
In  the  late  stages,  when  diagnosis  is  comparatively  easy, 
it  is  decreased.  Hence  estimation  of  urea  is  of  little  help 
in  the  diagnosis  of  this  disease,  especially  when,  as  is  so 
frequently  the  case,  a  small  quantity  of  urine  taken  at 
random  is  used.  When,  however,  the  diagnosis  is 
established,  estimations  made  at  frequent  intervals 
under  the  same  conditions  of  diet  and  exercise  are  of 
much  value,  provided  a  sample  of  the  mixed  twenty-four- 
hour  urine  he  used.  A  steady  decline  is  a  very  bad  prog- 
nostic sign,  and  a  sudden  marked  diminution  is  usually 
a  forerunner  of  uremia. 

The  presence  of  urea  can  be  shown  by  allowing  a  few 
drops  of  the  fluid  partially  to  evaporate  upon  a  slide,  and 
adding  a  small  drop  of  pure,  colorless  nitric  acid  or 
saturated  solution  of  oxalic  acid.  Crystals  of  urea 
nitrate  or  oxalate  (Fig.  23)  will  soon  appear  and  can  be 
recognized  with  the  microscope. 


94 


THE   URINE 


Quantitative  Estimation. — The  hypobromite  method, 
which  is  generally  used,  depends  upon  the  fact  that 
urea  is  decomposed  by  sodium  hypobromite  with  libera- 
tion of  nitrogen.     The  amount  of  urea  is  calculated  from 


Fig.  23. — Crystals  of  nitrate  of  urea  (upper  half)  and 
oxalate  of  urea  (lower  half)  (after  Funke). 


Fig.  24-- 


-Doremus-Hinds'  ure- 
ometer. 


the  volume  of  nitrogen  set  free.     The  improved  Doremus 
apparatus  (Fig.  24)  is  the  most  convenient. 

Pour  some  of  the  urine  into  the  smaller  tube  of  the  appa- 
ratus, then  open  the  stopcock  and  quickly  close  it  so  as  to  fill 
its  lumen  with  urine.  Rinse  out  the  larger  tube  with  water 
and  fill  it  and  the  bulb  with  25  per  cent,  caustic  soda  solu- 
tion. Add  to  this  i  c.c.  of  bromin  by  means  of  a  medicine- 
dropper  and  mix  well.  This  prepares  a  fresh  solution  of 
sodium  hypobromite  with  excess  of  caustic  soda,  which  serves 
to  absorb  the  carbon  dioxid  set  free  in  the  decomposition 
of  urea.  When  handling  bromin,  keep  an  open  vessel  of 
ammonia  near  to  neutralize  the  irritant  fumes. 

Pour  the  urine  into  the  smaller  tube,  and  then  turn  the 
stopcock  so  as  to  let  as  much  urine  as  desired  (usually  i  c.c.) 


CHEMIC  EXAMINATION  95 

run  slowly  into  the  hypobromite  solution.  When  bubbles 
have  ceased  to  rise,  read  off  the  height  of  the  fluid  in  the  large 
tube  by  the  graduations  upon  its  side.  This  gives  the  amount 
by  weight  of  urea  in  the  urine  added,  from  which  the  amount 
excreted  in  twenty-four  hours  can  easily  be  calculated.  If 
the  urine  contains  much  more  than  the  normal  amount,  it 
should  be  diluted. 

To  avoid  handling  pure  bromin,  which  is  disagreeable. 
Rice's  solutions  may  be  employed: 

(a)  Bromin,  31  gm. 
Potassium  bromid,  31 
Distilled  water,  250  c.c. 

(b)  Caustic  soda,  100  gm. 
Distilled  water,  250  c.c. 

One  part  of  each  of  these  solutions  and  two  parts  of  water 
are  mixed  and  used  for  the  test.  The  bromin  solution  must 
be  kept  in  a  tightly  stoppered  bottle  or  it  will  rapidly  lose 
strength. 

5.  Uric  Acid. — Uric  acid  is  the  most  important  of  a 
group  of  substances,  called  purin  bodies,  which  are  de- 
rived cliiefiy  from  the  nucleins  of  the  food  and  from 
metabolic  destruction  of  the  nuclei  of  the  body.  The 
daily  output  of  uric  acid  is  about  0.4  to  i  gm.  The 
amount  of  the  other  purin  bodies  together  is  about 
one-tenth  that  of  uric  acid.  Excretion  of  these  sub- 
stances is  greatly  increased  by  a  diet  rich  in  nucleins,  as 
sweetbreads  and  liver. 

Uric  acid  exists  in  the  urine  in  the  form  of  urates, 
which  in  concentrated  urines  are  readily  thrown  out  of 
solution  and  constitute  the  familiar  sediment  of  "  amor- 
phous urates."    This,  together  with  the  fact  that  uric 


96 


THE   URINE 


\.B9 
915 
2.45 


-fffl-s 


acid  is  frequently  deposited  as  crystals, 
constitutes  its  chief  interest  to  the  prac- 
titioner. It  is  a  very  common  error  to 
consider  these  deposits  as  evidence  of 
excessive  excretion. 

Pathologically,  the  greatest  increase 
of  uric  acid  occurs  in  leukemia,  where 
there  is  extensive  destruction  of  leuko- 
cytes, and  in  diseases  with  active  de- 
struction of  the  liver  and  other  organs 
rich  in  nuclei.  Uric  acid  is  decreased 
before  an  attack  of  gout  and  increased 
during  it,  but  its  etiologic  relation  is 
still  uncertain.  An  increase  is  also  noted 
in  acute  articular  rheumatism  during  the 
febrile  stage. 

Quantitative  Estimation. — The  follow- 
ing are  the  best  methods  for  ordinary 
clinical  purposes,  although  no  great  ac- 
curacy can  be  claimed  for  them. 

Cook's  Method  for  Purin  Bodies. — In  a 
centrifuge  tube  take  lo  c.c.  urine  and  add 
about  I  gm.  (about  i  c.c.)  sodium  car- 
bonate and  I  or  2  c.c.  strong  ammonia. 
Shake  until  the  soda  is  dissolved.  The 
earthy  phosphates  will  be  precipitated. 
Centrifugalize  thoroughly  and  pour  off  all 
the  clear  fluid  into  a  graduated  centrifuge 
tube.  Add  2  c.c.  ammonia  and  2  c.c.  am- 
moniated  silver  nitrate  solution.  Let  stand 
a  few  minutes,  and  revolve  in  the  centri- 
fuge until  the  bulk  of  precipitate  remains 
constant.    Each  one-tenth  cubic  centimeter 


CHEMIC  EXAMINATION  97 

of  sediment  represents  0.001176  gm.  purin  bodies.  This 
amount  may  be  regarded  as  uric  acid,  since  this  substance 
usually  constitutes  nine-tenths  of  the  purin  bodies  and  the 
clinical  significance  is  the  same. 

Ammoniated  silver  nitrate  solution  is  prepared  by  dissolving 
5  gm.  of  silver  nitrate  in  100  c.c.  distilled  water,  and  adding 
ammonia  until  the  solution  clouds  and  again  becomes  clear. 

Ruhemann's  Method  for  Uric  Acid. — The  urine  must 
be  slightly  acid.  Fill  Ruhemann's  tube  (Fig.  25)  to  the 
mark  S  with  the  indicator,  carbon  disulphid,  and  to  the  mark 
J  with  the  reagent.  The  carbon  disulphid  will  assume  a 
violet  color.  Add  the  urine,  a  small  quantity  at  a  time, 
closing  the  tube  with  the  glass  stopper  and  shaking  vigor- 
ously after  each  addition,  until  the  disulphid  loses  every 
trace  of  its  violet  color  and  becomes  pure  white.  This  com- 
pletes the  test.  The  figure  in  the  right-hand  column  of 
figures  corresponding  to  the  top  of  the  fluid  gives  the  amount 
of  uric  acid  in  parts  per  thousand.  The  presence  of  diacetic 
acid  interferes  with  the  test,  as  do  also,  to  some  extent,  bile 
and  albumin. 

Ruhemann's  reagent  consists  of  iodin  and  potassium  iodid 
each,  1.5  parts;  absolute  alcohol,  15  parts;  and  distilled  water, 
185  parts. 

6,  Ammonia. — A  small  amount  of  ammonia,  com- 
bined with  hydrochloric,  phosphoric,  and  sulphuric 
acids  is  always  present.  Estimated  as  NH3,  the  normal 
average  is  about  0.7  gm.  in  twenty-four  hours.  This 
represents  4  to  5  per  cent,  of  the  total  nitrogen  of  the 
urine,  ammonia  standing  next  to  urea  in  this  respect. 

Under  ordinary  conditions,  most  of  the  ammonia 
which  results  from  the  metabolic  processes  is  trans- 
formed into  urea.  When,  however,  acids  are  present 
in  excess,  either  from  ingestion  of  mineral  acids  or 
7 


98  THE   URINE 

from  abnormal  production  of  acids  within  the  body 
(as  in  fevers,  diabetes,  pernicious  vomiting  of  preg- 
nancy, etc.),  ammonia  combines  with  them  and  is  so 
excreted,  urea  being  correspondingly  decreased.  It  is 
thus  that  the  body  protects  itself  against  acid  intoxica- 
tion, A  marked  increase  of  ammonia  is,  therefore,  im- 
portant chiefly  as  an  index  of  the  tendency  to  acidosis, 
particularly  that  associated  with  the  presence  of  di- 
acetic  and  oxybutyric  acids. 

In  diabetes  mellitus  ammonia  elimination  may  reach 
4  or  5  gm.  daily.  It  is  likewise  markedly  increased  in 
pernicious  vomiting  of  pregnancy,  but  not  in  nervous 
vomiting;  and  in  conditions  in  which  the  power  to  syn- 
thesize urea  is  interfered  with,  notably  cirrhosis  and 
other  destructive  diseases  of  the  liver  and  conditions 
associated  with  deficient  oxygenation. 

Quantitative  Estimation. — The  urine  must  be  fresh, 
since  decomposition  increases  the  amount  of  ammonia. 
The  following  method  is  satisfactory  for  clinical  pur- 
poses, though  subject  to  some  inaccuracies. 

Ronchese-Malfatti  Formalin  Test. — This  depends  upon 
the  fact  that  when  formalin  is  added  to  the  urine,  the  am- 
monia combines  with  it,  forming  hexamethylene-tetramin. 
The  acids  with  which  the  ammonia  was  combined  are  set 
free,  and  their  quantity,  ascertained  by  titration  with  sodium 
hydroxid,  indicates  the  amount  of  ammonia. 

Take  10  c.c.  of  the  urine  in  a  beaker  or  evaporating  dish, 
add  50  c.c.  water  and  10  drops  of  0.5  per  cent,  alcoholic  solu- 
tion of  phenolphthaleln.  Neutralize  by  adding  a  weak 
caustic  soda  or  sodium  carbonate  solution  until  a  permanent 
pink  color  appears.  To  5  c.c.  formalin  add  15  c.c.  water 
and  neutralize  in  the  same  way.     Pour  the  formalin  into  the 


CHEMIC  EXAMINATION  99 

urine.  The  pink  color  at  once  disappears,  owing  to  libera- 
tion of  acids.  Now  add  decinormal  sodium  hydroxid  solution 
from  a  buret  until  the  pink  color  just  returns.  Each  cubic 
centimeter  of  the  decinormal  solution  used  in  this  titration 
corresponds  to  0.0017  E^-  of  NH3.  This  must  be  multi- 
plied by  ten  to  obtain  the  percentage  from  which  the  twenty- 
four-hour  elimination  of  ammonia  is  calculated. 

The  method  is  more  complicated,  but  distinctly  more 
accurate  when  carried  out  as  suggested  by  E.  W.  Brown. 
Treat  60  c.c.  of  urine  with  3  gm.  of  basic  lead  acetate,  stir 
well,  let  stand  a  few  minutes,  and  filter.  Treat  the  filtrate 
with  2  gm.  neutral  potassium  oxalate,  stir  well,  and  filter. 
Take  10  c.c.  of  the  filtrate,  add  50  c.c.  water  and  15  gm. 
neutral  potassium  oxalate,  and  proceed  with  the  ammonia 
estimation  as  above  outlined. 

B.    Abnormal  Constituents 

Those  substances  which  appear  in  the  urine  only  in 
pathologic  conditions  are  of  much  more  interest  to  the 
clinician  than  are  those  which  have  just  been  discussed. 
Among  them  are:  proteins,  sugars,  the  acetone  bodies, 
bile,  hemoglobin,  and  the  diazo  substances.  The  "  pan- 
creatic reaction  "  and  detection  of  drugs  in  the  urine  will 
also  be  discussed  under  this  head. 

I.  Proteins. — Of  the  proteins  which  may  appear 
in  the  urine,  serum-albumin  and  serum-globulin  are  the 
most  important.  Mucin,  proteose,  and  a  few  others  are 
found  occasionally,  but  are  of  less  interest. 

(i)  Serum-albumin  and  Serum-globulin. — These  two 
proteins  constitute  the  so-called  "  urinary  albumin." 
They  usually  occur  together,  have  practically  the  same 
significance,  and  both  respond  to  all  the  ordinary  tests 
for  "  albumin." 


lOO  THE   URINE 

Their  presence,  or  albuminuria,  is  probably  the  most 
important  pathologic  condition  of  the  urine.  It  is 
either  accidental  or  renal.  The  physician  can  make  no 
greater  mistake  than  to  regard  all  cases  of  albuminuria 
as  indicating  kidney  disease. 

Accidental  or  Jalse  albuminuria  is  due  to  admixture 
with  the  urine  of  albuminous  fluids,  such  as  pus,  blood, 
and  vaginal  discharge.  The  microscope  will  usually 
reveal  its  nature.  It  occurs  most  frequently  in  pyelitis, 
cystitis,  and  chronic  vaginitis. 

Renal  albuminuria  refers  to  albumin  which  has  passed 
from  the  blood  into  the  urine  through  the  walls  of  the 
kidney  tubules  or  the  glomeruli. 

Albuminuria  sufficient  to  be  recognized  by  clinical 
methods  probably  never  occurs  as  a  physiologic  condi- 
tion, the  so-called  physiologic  albuminuria  appearing 
only  under  conditions  which  must  be  regarded  as 
abnormal.  Among  these  may  be  mentioned  excessive 
muscular  exertion  in  those  unaccustomed  to  it;  exces- 
sive ingestion  of  proteins;  prolonged  cold  baths;  and 
childbirth.  In  these  conditions  the  albuminuria  is 
slight  and  transient. 

There  are  certain  other  forms  of  albuminuria  which 
have  still  less  claim  to  be  called  physiologic,  but  which 
are  not  always  regarded  as  pathologic.  Among  these 
are  cyclic  albuminuria,  which  regularly  recurs  at  a 
certain  period  of  the  day,  and  orthostatic  or  postural 
albuminuria,  which  appears  only  when  the  patient  is 
standing.  They  are  rare  and  of  obscure  origin,  and 
occur  for  the  most  part  in  neurasthenic  subjects  during 
adolescence.  It  is  noteworthy  in  this  connection  that 
nephritis  sometimes  begins  with  a  cycUc  albuminuria. 


CHEMIC  EXAMINATION     f-fRpApwlOr 

""'^^f  I   rrr-  *   ^^ 

In  pathologic  conditions  and  in  most,  at  least/  of'liE^  r/I  "j 
"functional"    conditions   just   enumerated,    renal  D^f.  n  f 
buminuria  may  be  referred  to  one  or  more  of  the  follow- 
ing causes.     In  nearly  all  cases  it  is  accompanied  by 
tube-casts. 

(a)  Changes  in  the  blood  which  render  its  albumin 
more  diffusible,  as  in  severe  anemias,  purpura,  and 
scurvy.    Here  the  albumin  is  small  in  amount. 

(Z>)  Changes  in  circulation  in  the  kidney,  either  anemia 
or  congestion,  as  in  excessive  exercise,  chronic  heart 
disease,  and  pressure  upon  the  renal  veins.  The  quan- 
tity of  albumin  is  usually,  but  not  always,  small.  Its 
presence  is  constant  or  temporary,  according  to  the 
cause.  Most  of  the  causes,  if  continued,  will  produce 
organic  changes  in  the  kidney. 

(c)  Organic  Changes  in  the  Kidney. — These  include 
the  inflammatory  and  degenerative  changes  commonly 
grouped  together  under  the  name  of  nephritis,  and  also 
renal  tuberculosis,  neoplasms,  and  cloudy  swelling  due 
to  irritation  of  toxins  and  drugs.  The  amount  of  al- 
bumin eliminated  in  these  conditions  varies  from  minute 
traces  to  20  gm.,  or  even  more,  in  the  twenty-four  hours, 
and,  except  in  acute  processes,  bears  little  relation  to 
the  severity  of  the  disease.  In  acute  and  chronic 
parenchymatous  nephritis  the  quantity  is  usually  very 
large.  In  chronic  interstitial  nephritis  it  is  small — 
frequently  no  more  than  a  trace.  It  is  small  in  cloudy 
swelling  from  toxins  and  drugs,  and  variable  in  renal 
tuberculosis  and  neoplasms.  In  amyloid  disease  of  the 
kidney  the  quantity  is  usually  small,  and  serum-globulin 
may  be  present  in  especially  large  proportion,  or  even 
alone.     Roughly   distinctive   of   serum-globulin   is   the 


I02  THE   URINE 

appearance  of  an  opalescent  cloud  when  a  few  drops  of 
the  urine  are  dropped  into  a  glass  of  distilled  water. 

Detection  of  albumin  depends  upon  its  precipitation 
by  chemicals  or  coagulation  by  heat.  There  are  many 
tests,  but  none  is  entirely  satisfactory,  because  other 
substances  as  well  as  albumin  are  precipitated.  The 
most  common  source  of  error  is  mucin.  The  tests  given 
here  are  widely  used  and  can  be  recommended.  They 
make  no  distinction  between  serum-albumin  and  serum- 
globulin.  They  are  given  as  nearly  as  possible  in  order 
of  their  delicacy.  Usually  the  best  time  to  detect 
albumin  is  in  the  evening  or  a  few  hours  after  a  meal. 

//  is  very  important  that  urine  to  he  tested  for  albumin 
he  rendered  clear  hy  filtration  or  centrifugation.  This 
is  too  often  neglected  in  routine  work.  When  ordinary 
methods  do  not  suffice,  it  can  usually  be  cleared  by 
shaking  up  with  a  little  purified  talc  or  animal  charcoal 
and  filtering. 

(i)  Trichloracetic  Acid  Test. — The  reagent  consists  of  a 
saturated  aqueous  solution  of  trichloracetic  acid  to  which 
magnesium  sulphate  is  added  to  saturation.  A  simple 
saturated  solution  of  the  acid  may  be  used,  but  addition  of 
magnesium  sulphate  favors  precipitation  of  globulin,  and, 
by  raising  the  specific  gravity,  makes  the  test  easier  to 
apply. 

Take  a  few  cubic  centimeters  of  the  reagent  in  a  test-tube 
or  conical  test  glass,  hold  the  tube  or  glass  in  an  inclined 
position,  and  run  the  urine  gently  in  by  means  of  a  pipet,  so 
that  it  will  form  a  layer  on  top  of  the  reagent  without  mix- 
ing with  it.  If  albumin  be  present,  a  white,  cloudy  ring  will 
appear  where  the  two  fluids  come  in  contact.  The  ring  can 
be  seen  most  clearly  if  viewed  against  a  black  background, 


CHEMIC  EXAMINATION  I03 

and  one  side  of  the  tube  or  conical  glass  may  be  painted  black 
for  this  purpose. 

This  is  an  extremely  sensitive  test,  but,  unfortunately, 
both  mucin  and  proteoses  respond  to  it;  urates,  when  abund- 
ant, may  give  a  confusing  white  ring,  and  the  reagent  is  com- 
paratively expensive.  It  is  not  much  used  in  routine  work 
except  as  a  control  to  the  less  sensitive  tests. 


Fig.  26. — Horismascope:  adding  the  reagent. 

A  most  convenient  instrument  for  applying  this  or  any  of 
the  contact  tests  is  sold  under  the  name  of  "  horismascope  " 
(Fig.  26). 

(2)  Robert's  Test. — ^The  reagent  consists  of  pure  nitric 
acid,  I  part,  and  saturated  aqueous  solution  of  magnesium 
sulphate,  5  parts.  It  is  applied  in  the  same  way  as  the  pre- 
ceding test. 

Albumin  gives  a  white  ring,  which  varies  in  density  with 


I04  THE   URINE 

the  amount  present.  A  similar  white  ring  may  be  produced 
by  primary  proteose  and  resinous  drugs.  White  rings  or 
cloudiness  in  the  urine  above  the  zone  of  contact  may  result 
from  excess  of  urates  or  mucus.  Colored  rings  near  the  junc- 
tion of  the  fluids  may  be  produced  by  urinary  pigments, 
bile,  or  indicari. 

Robert's  test  is  one  of  the  best  for  routine  work,  although 
the  various  rings  are  apt  to  be  confusing  to  the  inexperienced. 
It  is  more  sensitive  than  Heller's  test,  of  which  it  is  a  modifica- 
tion, and  has  the  additional  advantage  that  the  reagent  is 
not  so  corrosive. 

(3)  Purdy's  Heat  Test. — Take  a  test-tube  two-thirds  full 
of  urine,  add  about  one-sixth  its  volume  of  saturated  solution 
of  sodium  chlorid,  and  5  to  10  drops  of  50  percent,  acetic  acid. 
Mix,  and  boil  the  upper  inch.  A  white  cloud  in  the  heated 
portion  shows  the  presence  of  albumin. 

This  is  a  valuable  test  for  routine  work.  It  is  simple, 
sufficiently  accurate  for  clinical  purposes,  and  has  practically 
no  fallacies.  Addition  of  the  salt  solution,  by  raising  the 
specific  gravity,  prevents  precipitation  of  mucin.  Proteose 
may  produce  a  white  cloud,  which  disappears  upon  boiling 
and  reappears  upon  cooling. 

(4)  Heat  and  Nitric  Acid  Test. — This  is  one  of  the  oldest 
of  the  albumin  tests,  and  if  properly  carried  out,  one  of  the 
best.  Boil  a  small  quantity  of  filtered  urine  in  a  test-tube  and 
add  about  one-twentieth  its  volume  of  concentrated  nitric 
acid.  A  white  cloud  or  flocculent  precipitate  (which  usually 
appears  during  the  boiling,  but  if  the  quantity  be  very  small 
only  after  addition  of  the  acid)  denotes  the  presence  of  albu- 
min. A  similar  white  precipitate,  which  disappears  upon 
addition  of  the  acid,  is  due  to  earthy  phosphates.  The  acid 
should  not  be  added  before  boiling,  and  the  proper  amount 
should  always  be  used;  otherwise,  part  of  the  albumin  may 
fail  to  be  precipitated  or  may  be  redissolved. 


CHEMIC  EXAMINATION 


105 


Quantitative  Estimation. — The  gravimetric,  which  is 
the  most  reliable  method,  is  too  elaborate  for  clinical 
work.  Both  Esbach's,  which  is  very  widely  used,  and 
the  centrifugal  method  give  fair  results,  but  Tsuchiya's 
recent  modification  of  the  Esbach 
method  is  preferable  to  either. 

(i)  Esbach's  Method. — ^The  urine  must 
be  clear,  of  acid  reaction,  and  not  con- 
centrated. Always  filter  before  testing, 
and,  if  necessary,  add  acetic  acid  and  dilute 
with  water.  Esbach's  tube  (Fig.  27)  is  es- 
sentially a  test-tube  with  a  mark  U  near 
the  middle,  a  mark  R  near  the  top,  and 
graduations  J,  i,  2,  3,  etc.,  near  the  bot- 
tom. Fill  the  tube  to  the  mark  U  with 
urine  and  to  the  mark  R  with  the  reagent. 
Close  with  a  rubber  stopper,  invert  slowly 
several  times,  and  set  aside  in  a  cool  place. 
At  the  end  of  twenty-four  hours  read  off 
the  height  of  the  precipitate.  This  gives 
the  amount  of  albumin  in  grams  per  liter, 
and  must  be  divided  by  10  to  obtain  the 
percentage. 

Esbach's  reagent  consists  of  picric  acid,  i 
2  gm.,  and  distilled  water,  to  make  100  c.c. 

(2)  Tsuchiya's  Method. — ^This  is  carried  out  in  the  same 
manner  as  the  Esbach  method,  using  the  following  reagent: 

Phosphotungstic  acid 1.5  gm. 

96  per  cent,  alcohol 95.0  c.c. 

Concentrated  hydrochloric  acid 5-o    " 

The  urine  should  be  diluted  to  a  specific  gravity  not  exceeding 
1.008.    The  method  is  said  to  be  much  more  accurate  than 


Fig.  27. — Esbach's 
albuminometer,  im- 
proved form. 

gm.,  citric  acid, 


I06  THE    URINE 

the  original  Esbach  method,  particularly  with  small  quanti- 
ties of  albumin. 

(3)  Purdy's  Centrifugal  Method. — This  is  detailed  in 
the  table  on  opposite  page.  The  percentage  by  weight  is 
approximately  one-fiftieth  of  the  bulk  percentage. 

(2)  Mucin. — Traces  of  the  substances  (mucin,  mu- 
coid, etc.)  which  are  loosely  classed  under  this  name  are 
present  in  normal  urine;  increased  amounts  are  observed 
in  irritations  and  inflammations  of  the  mucous  mem- 
brane of  the  urinary  tract.  They  are  of  interest  chiefly 
because  they  may  be  mistaken  for  albumin  in  most  of 
the  tests.  If  the  urine  be  diluted  with  water  and  acidi- 
fied with  acetic  acid,  the  appearance  of  a  w'hite  cloud  in- 
dicates the  presence  of  mucin. 

True  mucin  is  a  glyco-protein,  and  upon  boiling  with 
an  acid  or  alkali,  as  in  Fchling's  test,  yields  a  carbohy- 
drate substance  which  reduces  copper. 

(3)  Proteoses. — These  are  intermediate  products  in 
the  digestion  of  proteins  and  are  frequently,  although 
incorrectly,  called  albumoses.  Tw'O  groups  are  generally 
recognized:  primary  proteoses,  w^hich  are  precipitated 
upon  half-saturation  of  their  solutions  wdth  ammonium 
sulphate;  and  secondary  proteoses,  which  are  precipitated 
only  upon  complete  saturation. 

The  secondary  proteoses  have  been  observed  in  the 
urine  in  febrile  and  malignant  diseases  and  chronic  sup- 
purations, during  resolution  of  pneumonia,  and  in  many 
other  conditions,  but  their  clinical  significance  is  in- 
definite. In  pregnancy,  albumosuria  may  be  due  to 
absorption  of  amniotic  fluid. 

Primary  proteoses  are  rarely  encountered  in  the  urine. 


CHEMIC  EXAMINATION 


107 


PURDY'S    QUANTITATIVE    METHOD    FOR   ALBUMIN    IN 
URINE    (CENTRIFUGAL). 

Table  showing  the  relation  between  the  volumetric  and  gravimetric  percentage 

of  albumin  obtained  by  means  of  the  centrifuge  with  radius  of  six 

and  three-quarter  inches  ;  rate  of  speed,  i$oo  revolutions 

per  minute  ;    time,  three  minutes. 


> 

> 

> 

>*     ^ 

> 

> 

^mM 

>2 

d!  W  S 

u"  (li 

"h2 

a/  til  2 

H>' 

"fc2 

«  w  2 

•J  u  2 

0  n!  u 

r>WU 
Oh 

w  0  s 

<  H  5 

(1,    Q 

(U  Z  ;-) 

III 
III 

m 

3  H  i- 

D  W  h 
J  U  z 
p  «  W 

Pi 

•^  £  D 

D  w  H 
►J  UZ 
p  «  W 
>WCJ 

w  0  s 

5  t  n 
fc  K  J 

^>^ 

K 

0.005 

0.025 

135^ 

0.281 

1-35 

3iJ4 

0.656 

3-15 

Vt 

O.OI 

0.05 

14 

0.292 

1.4 

32 

0.667 

3-2 

K 

0.016 

0.075 

14^ 

0.302 

145 

32^ 

0.677 

3-25 

I 

0.021 

0.1 

^5  , 

0-313 

1-5 

33 

0.687 

3-3 

'K 

0.026 

0.125 

15^ 

0.323 

1-55 

3354 

0.698 

3-35 

IM 

0.031 

0.15 

16 

0-333 

1.6 

34 

0.708 

3-4 

iK 

0.036 

0.175 

16^ 

0.344 

1.65 

3454 

0.719 

3-45 

2 

0.042 

0.2 

17 

0.354 

1-7 

35 

0.729 

3-5 

2^ 

0.047 

0.225 

17^ 

0-365 

1-75 

3554 

0.74 

3-55 

^% 

0.052 

0.25 

18 

0.375 

1.8 

36 

0-75 

3-6 

iV* 

0.057 

0.275 

18'^ 

0-385 

1-85 

3654 

0.76 

3-65 

3 

0.063 

0-3 

19 

0.396 

1-9 

37  , 

0.771 

3-7 

3K 

0.068 

0.325 

19H 

0.406 

1-95 

3754 

0.781 

3-75 

Z'A 

0.073 

0.35 

20 

0.417 

2. 

38 

0.792 

3.8 

1% 

0.078 

0-375 

rtoV, 

0.427 

2.05 

385^ 

0.801 

3.85 

4 

0.083 

0.4 

21 

0.438 

2.1 

39 

0.813 

3-9 

4^ 

0.089 

0.425 

2154 

0.448 

2.15 

395^ 

0.823 

3-95 

4'/^ 

0.094 

0-45 

22 

0.458 

2.2 

40 

0-833 

4- 

4K 

0.099 

0-475 

22^ 

0.469 

2.25 

4054 

0.844 

4-05 

5 

0.104 

0-5 

23 

0-479 

2.3 

41  , 

0.854 

4-1 

55^ 

O.III 

0-55 

23J4 

0-49 

2-35 

4154 

0.865 

4-15 

6 

0.125 

0.6 

24 

0.5 

2-4 

42 

0.875 

4-2 

654 

0.135 

0.65 

24  J^ 

0.51 

2-45 

4254 

0.885 

4-25 

7 

0.146 

0.7 

25 

0.521 

2-5 

43  , 

0.896 

4-3 

7H 

0.156 

0-75 

25^ 

0-531 

2.55 

4354 

0.906 

4-35 

8 

0.167 

0.8 

26 

0-542 

2.6 

44 

0.917 

4-4 

8}^ 

0.177 

0.85 

26J4 

0-552 

2.65 

4454 

0.927 

4-45 

9 

0.187 

0.9 

27 

0-563 

2.7 

45  , 

0.938 

4-5 

9J4 

0.198 

0-95 

21% 

0-573 

2.75 

4554 

0.948 

4-55 

10 

0.208 

1. 

28 

0-583 

2.8 

46 

0.958 

4.6 

1054 

0.219 

1.05 

28J^ 

0-594 

2.85 

4654 

0.969 

4-65 

II 

0.229 

i.l 

29 

0.604 

2-9 

47  , 

0-979 

4-7 

iiH 

0.24 

I-I5 

29  J^ 

0.615 

2-95 

4754 

0.99 

4-75 

12 

0.25 

1.2 

30 

0.625 

3- 

48 

I. 

4-8 

1254 

0.26 

125 

30^ 

0.635 

3-05 

13 

0.271 

1-3 

31 

0.646 

3-1 

Test. — Three  cubic  centimeters  of  10  per  cent,  solution  of  ferrocyanid  of 
potassium  and  2  cubic  centimeters  of  50  per  cent,  acetic  acid  are  added  to  10  cubic 
centimeters  of  the  urine  in  the  percentage  tube  and  stood  aside  for  ten  minutes, 
then  placed  in  the  centrifuge  and  revolved  at  rate  of  speed  and  time  as  stated  at 
head  of  the  table.  If  albumin  is  excessive,  dilute  the  urine  with  water  until 
volume  of  albumin  falls  below  10  per  cent.  Multiply  result  by  the  number 
of  dilutions  employed  before  using  the  table. 


Io8  THE   URINE 

The  protein  known  as  the  "  Bence- Jones  body  "  was 
originally  classed  under  this  head,  but  its  true  nature  is 
uncertain.  It  is  regarded  as  practically  pathognomonic 
of  multiple  myeloma. 

The  proteoses  are  not  coagulable  by  heat,  but  are  precipi- 
tated by  such  substances  as  trichloracetic  acid  and  phos- 
photungstic  acid.  The  primary  proteoses,  alone,  are  pre- 
cipitated by  nitric  acid. 

Proteoses  may  be  detected  by  acidifying  the  urine  with 
acetic  acid,  boiling  and  filtering  while  hot  to  remove  mucin, 
albumin,  and  globulin,  and  testing  the  filtrate  by  the  tri- 
chloracetic acid  test.  As  above  indicated,  the  nitric  acid 
test,  and  half  and  complete  saturation  with  ammonium  sul- 
phate will  separate  the  two  groups. 

To  detect  Bence- Jones'  body  the  urine  is  acidified  with 
acetic  acid  and  gently  heated.  If  this  substance  be  present, 
a  precipitate  will  form  at  about  60°  C.  As  the  boiling-point 
is  reached,  it  wholly  or  partially  dissolves.  It  reappears 
upon  cooling. 

2.  Sugars. — Various  sugars  may  at  times  be  found  in 
the  urine.  Dextrose  is  by  far  the  most  common,  and  is 
the  only  one  of  clinical  importance.  Levulose,  lactose, 
and  some  others  are  occasionally  met  with. 

(i)  Dextrose  (Glucose). — It  is  probable  that  traces  of 
glucose,  too  small  to  respond  to  the  ordinary  tests,  are 
present  in  the  urine  in  health.  Its  presence  in  appre- 
ciable amount  constitutes  "  glycosuria." 

Transitory  glycosuria  is  unimportant,  and  may  occur 
in  many  conditions,  as  after  general  anesthesia  and 
administration  of  certain  drugs,  in  pregnancy,  and 
following  shock  and  head  injuries.     It  may  also  occur 


CHEMIC   EXAMINATION  I09 

after  eating  excessive  amounts  of  carbohydrates  (ali- 
mentary glycosuria). 

Persistent  glycosuria  has  been  noted  in  brain  injuries 
involving  the  floor  of  the  fourth  ventricle.  As  a  rule, 
however,  persistent  glycosuria  is  diagnostic  of  diabetes 
mellitus,  of  which  disease  it  is  the  essential  symptom. 
The  amount  of  glucose  eliminated  in  diabetes  is  usually 
considerable,  and  is  sometimes  very  large,  reaching  500 
gm.,  or  even  more,  in  twenty-four  hours,  but  it  does  not 
bear  any  uniform  relation  to  the  severity  of  the  disease. 
Glucose  may,  on  the  other  hand,  be  almost  or  entirely 
absent  temporarily. 

Detection  of  Dextrose. — If  albumin  be  present  in  more 
than  traces,  it  must  be  removed  by  boiling  and  filtering. 

(i)  Haines*  Test. — Take  about  i  dram  of  Haines'  solution 
in  a  test-tube,  boil,  and  add  6  or  8  drops  of  urine.  A  heavy 
yellow  or  red  precipitate,  which  settles  readily  to  the  bottom, 
shows  the  presence  of  sugar.  Neither  precipitation  of  phos- 
phates as  a  light,  flocculent  sediment  nor  simple  decolorization 
of  the  reagent  should  be  mistaken  for  a  positive  reaction. 

This  is  one  of  the  best  of  the  copper  tests,  all  of  which 
depend  upon  the  fact  that  in  strongly  alkaline  solutions  glucose 
reduces  cupric  hydrate  to  cuprous  hydrate  (yellow)  or  cup- 
rous oxid  (red).  They  are  somewhat  inaccurate,  because 
they  make  no  distinction  between  glucose  and  less  common 
forms  of  sugar;  because  certain  normal  substances,  when 
present  in  excess,  especially  mucin,  uric  acid,  and  creatinin, 
may  reduce  copper,  and  because  many  drugs — e.  g.,  chloral, 
-chloroform,  copaiba,  acetanilid,  benzoic  acid,  morphin, 
sulphonal,  salicylates — are  eliminated  as  copper-reducing 
substances.  To  minimize  these  fallacies  dilute  the  urine,  if 
it  be  concentrated;  do  not  add  more  than  the  specified  amount 
of  urine,  and  do  not  boil  after  the  urine  is  added. 


no 


THE    URINE 


Haines''  solution  is  prepared  as  follows:  completely  dissolve 
30  gr.  pure  copper  sulphate  in  \  oz.  distilled  water,  and  add 
2  oz.  pure  glycerin;  mix  thoroughly,  and  add  5  oz.  liquor 
potassae.     The  solution  keeps  well. 

(2)  Fehling's  Test. — Two  solutions  are  required — one 
containing  34.64  gm.  pure  crystalline  copper  sulphate  in 
500  c.c.  distilled  water;  the  other,  173  gm.  Rochelle  salt  and 
100  gm.  potassium  hydroxid  in  500' c.c.  distilled  water.  Mix 
equal  parts  of  the  two  solutions  in  a  test-tube,  dilute  with 


Fig.  28. — Crystals  of  phenylclucosazone  from  diabetic  urine — Kowarsky's  test  (  X  500). 

3  or  4  volumes  of  water,  and  boil.  Add  the  urine  a  little  at  a 
time,  heating,  but  not  boiling,  between  additions.  In  the 
presence  of  glucose  a  heavy  red  or  yellow  precipitate  will 
appear.  The  quantity  of  urine  should  not  exceed  that  of  the 
reagent. 

(3)  Benedict's  Test. — This  new  test  promises  to  displace 
all  other  reduction  tests  for  glucose.  The  reagent  is  said  to 
be  ten  times  as  sensitive  as  Haines'  or  Fehling's,  and  not  to 
be  reduced  by  uric  acid,  creatinin,  chloroform,  or  the  alde- 
hyds.     It  consists  of: 


CHEMIC  EXAMINATION  III 


17-3  gm. 

173-0    " 

200.0     " 

lOOO.O  c.c. 

Copper  sulphate  (pure  crystallized), 
Sodium  or  potassium  citrate, 
Sodium  carbonate  (crystallized), 

(or  IOC  gm.  of  the  anhydrous  salt). 
Distilled  water,  to  make 


Dissolve  the  citrate  and  carbonate  in  700  c.c.  of  water,  with 
the  aid  of  heat,  and  filter.  Dissolve  the  copper  in  100  c.c. 
of  water  and  pour  slowly  into  the  first  solution,  stirring  con- 
stantly. Cool,  and  make  up  to  one  liter.  The  reagent  keeps 
indefinitely. 

Take  about  5  c.c.  of  this  reagent  in  a  test-tube,  and  add 
8  or  10  drops  {not  more)  of  the  urine.  Heat  to  vigorous 
boiling,  keep  at  this  temperature  for  one  or  two  minutes, 
and  allow  to  cool  slowly.  In  the  presence  of  glucose  the 
entire  body  of  the  solution  will  be  filled  with  a  precipitate, 
which  may  be  red,  yellow,  or  green  in  color.  When  traces 
only  of  glucose  are  present,  the  precipitate  may  appear  only, 
upon  cooling.  In  the  absence  of  glucose,  the  solution  re- 
mains clear  or  shows  only  a  faint,  bluish  precipitate,  due  to 
urates. 

(4)  Phenylhydrazin  Test. —  Kowarsky^s  Method. — In  a  wide 
test-tube  take  5  drops  pure  phenylhydrazin,  10  drops  glacial 
acetic  acid,  and  i  c.c.  saturated  solution  of  sodium  chlorid. 
A  curdy  mass  results.  Add  2  or  3  c.c.  urine,  boil  for  at  least 
two  minutes,  and  set  aside  to  cool.  Examine  the  sediment 
with  the  microscope,  using  a  two-thirds  objective.  If  glucose 
be  present,  characteristic  crystals  of  phenylglucosazone  will 
be  seen.  These  are  yellow,  needle-like  crystals  arranged 
mostly  in  clusters  or  in  sheaves  (Fig.  28).  When  traces  only 
of  glucose  are  present,  the  crystals  may  not  appear  for  one- 
half  hour  or  more.  Best  crystals  are  obtained  when  the  fluid 
is  cooled  very  slowly.  It  must  not  be  agitated  during 
cooling. 


112  THE    URINE 

This  is  an  excellent  test  for  clinical  work.  It  requires 
slightly  more  time  than  Haines'  test,  but  more  than  compen- 
sates for  this  by  increased  accuracy.  It  is  fully  as  sensitive 
as  Haines',  and  has  practically  no  fallacies  excepting  levulose, 
which  is  a  fallacy  for  all  tests  but  the  polariscope.  Other 
carbohydrates  which  are  capable  of  forming  crystals  with 
phenylhydrazin  are  extremely  imlikely  to  do  so  when  the  test 
is  appUed  directly  to  the  urine  by  the  method  just  detailed. 
Even  if  not  used  routinely,  this  test  should  always  be  resorted 
to  when  Haines'  test  gives  a  positive  reaction  in  doubtful 
cases. 

Quantitative  Estimation. — In  quantitative  work  Feh- 
ling's  solution,  for  so  many  years  the  standard,  has  been 
largely  displaced  by  Purdy's,  which  avoids  the  heavy 
precipitate  that  so  greatly  obscures  the  end-reaction  in 
Fehling's  method.  The  older  method  is  still  preferred 
by  many,  and  both  are,  therefore,  given.  The  new 
method  of  Benedict  is  likewise  included,  since  it  appears 
to  be  more  exact  than  any  other  titration  method 
available  for  sugar  work.  Should  the  urine  contain 
much  glucose,  it  must  be  diluted  before  making 
any  quantitative  test,  allowance  being  made  for  the 
dilution  in  the  subsequent  calculation.  Albumin,  if 
present,  must  be  removed  by  acidifying  a  considerable 
quantity  of  urine  with  acetic  acid,  boiling,  and  filtering. 
The  precipitate  should  then  be  washed  with  water  and 
the  washings  added  to  the  urine  to  bring  it  to  its  original 
volume. 

(i)  Purdy's  Method. — Take  exactly  35  c.c.  of  Purdy's 
solution  in  a  flask  or  beaker,  add  twice  its  volume  of  distilled 
water,  heat  to  boiling,  and,  still  keeping  the  solution  hot,  add 


CHEMIC  EXAMINATION  II3 

the  urine  very  slowly  from  a  buret  until  the  blue  color  entirely 
disappears.  Read  off  the  amount  of  urine  added;  considering 
the  strength  of  Purdy's  solution,  it  is  readily  seen  that  this 
amount  of  urine  contains  0.02  gm.  of  glucose,  from  which  the 
amount  in  the  twenty-four-hour  urine,  or  the  percentage,  can 
easily  be  calculated.  Example:  Suppose  that  2.5  c.c.  of 
urine  discharged  the  blue  color  of  35  c.c.  of  Purdy's  solution. 
This  amount  of  urine,  therefore,  contains  exactly  0.02  gm. 
glucose,  and  the  percentage  is  obtained  from  the  equation: 
2.5  :  100  :  :  0.02  :  x,  and  x  equals  0.8  per  cent.  If,  then, 
the  twenty-four-hour  quantity  of  urine  were  3000  c.c,  the 
twenty-four-hour  elimination  of  glucose  would  be  found  as 
follows:  100  :  3000  :  :  0.8  :  x,  and  x  equals  24  gm. 

It  will  be  found  that  after  the  test  is  completed  the  blue 
color  slowly  returns.  This  is  due  to  reoxidation,  and  should 
not  be  mistaken  for  incomplete  reduction. 

A  somewhat  simpler  application  of  this  method,  which  is 
accurate  enough  for  clinical  purposes,  is  as  follows:  Take 
84  c.c.  (roughly,  9  c.c.)  of  Purdy's  solution  in  a  large  test- 
tube,  dilute  with  an  equal  volume  of  water,  heat  to  boiling, 
and,  while  keeping  the  solution  hot  but  not  boUing,  add  the 
urine  drop  by  drop  from  a  medicine-dropper  until  the  blue 
color  is  entirely  gone.  Toward  the  end  add  the  drops  very 
slowly,  not  more  than  4  or  5  a  minute.  Divide  10  by  the 
number  of  drops  required  to  discharge  the  blue  color;  the 
quotient  will  be  the  percentage  of  glucose.  If  20  drops  were 
required,  the  percentage  would  be  10-^-20  =  0.5  P^^  cent. 
It  is  imperative  that  the  drops  be  of  such  size  that  20  of  them 
will  make  i  c.c.  Test  the  dropper  with  urine,  not  water.  If 
the  drops  are  too  large,  draw  out  the  tip  of  the  dropper;  if 
too  small,  file  off  the  tip. 

Ptirdy's  solution  consists  of  pure  crystalline  copper  sulphate, 
4.752  gm. ;  potassium  hydroxid,  23.5  gm.;  ammonia  (U.  S.  P.; 
sp.  gr.,  0.9),  350  c.c. ;  glycerin,  7,8  c.c. ;  distilled  water,  to  make 
8 


114 


THE   URINE 


looo  c.c.  Dissolve  the  copper  sulphate  and  glycerin  in  200 
c.c.  of  the  water  by  aid  of  gentle  heat.  In  another  200  c.c. 
pf  water  dissolve  the  potassium  hydroxid.  Mix  the  two  solu- 
tions, and  when  cool,  add  the  ammonia.  Lastly,  bring  the 
whole  up  to  1000  c.c.  with  distilled  water.  This  solution  is 
pf  such  strength  that  the  copper  in  35  c.c.  will  be  reduced  by 
exactly  0.02  gm.  of  glucose. 

(2)  Fehling's  Method. — Take  10  c.c.  Fehling's  solution 
(made  by  mixing  5  c.c.  each  of  the  copper  and  alkaline  solu- 
tions described  on  page  no)  in  a  flask  or  beaker,  add  three  or 


Fig.  29. — Einhorn's  saccharimeter. 


four  volumes  of  water,  boil,  and  add  the  urine  very  slowly  from 
a  buret  until  the  solution  is  completely  decolorized,  heating 
but  not  boiling  after  each  addition. 

The  chief  objection  to  Fehling's  method  is  the  difficulty 
of  determining  the  end-point.  The  use  of  an  "  outside  indi- 
cator," however,  obviates  this.  When  reduction  is  thought 
to  be  complete,  a  few  drops  of  the  solution  are  filtered  through 
a  fine-grained  filter-paper  on  to  a  porcelain  plate,  quickly 
acidified  with  acetic  acid,  and  mixed  with  a  drop  of  10  per 


CHEMIC  EXAMINATION  II5 

cent,  potassium  ferrocyanid.      Immediate  appearance  of  a 
red-brown  color  shows  the  presence  of  unreduced  copper. 

Fehling's  solution  is  of  such  strength  that  the  copper  in 
10  c.c.  will  be  reduced  by  exactly  0.05  gm.  of  glucose.  There- 
fore, the  amount  of  urine  required  to  decolorize  the  test  solu- 
tion contains  just  0.05  gm.  glucose,  and  the  percentage  is 
easily  calculated. 

(3)  Benedict's  Method. — The  following  modification  of 
his  copper  solution  has  recently  been  offered  by  Benedict 
for  quantitative  estimations. 

The  reagent  consists  of: 

Copper  sulphate  (pure  crystallized),  18.0  gm. 

Sodium  carbonate  (crystallized),  200.0    " 

(or  100  gm.  of  the  anhydrous  salt). 

Sodium  or  potassium  citrate,  200.0    " 

Potassium  sulphocyanate,  125.0    '* 
5  per  cent,  potassium  ferrocyanid  solution,      5.0  c.c. 

Distilled  water,  to  make  looo.o    " 

With  the  aid  of  heat  dissolve  the  carbonate,  citrate,  and 
sulphocyanate  in  about  800  c.c.  of  the  water  and  filter. 
Dissolve  the  copper  in  100  c.c.  of  water  and  pour  slowly 
into  the  other  fluid,  stirring  constantly.  Add  the  ferro- 
cyanid solution,  cool,  and  dilute  to  1000  c.c.  Only  the 
copper  need  be  accurately  weighed.  This  solution  is  of  such 
strength  that  25  c.c.  are  reduced  by  0.05  gram  glucose.  It 
keeps  well. 

To  make  a  sugar  estimation,  take  25  c.c.  of  the  reagent  in 
a  porcelain  evaporating  dish,  add  10  to  20  grams  sodium 
.carbonate  crystals  (or  one-half  this  weight  of  the  anhydrous 
salt)  and  a  small  quantity  of  powdered  pumice-stone  or  tal- 
cum. Heat  to  boiling,  and  add  the  urine  rather  rapidly 
from  a  buret  until  a  chalk-white  precipitate  forms  and 
the  blue  color  of  the  reagent  begins  to  fade.     After  this 


Il6  THE    URINE 

point  is  reached,  add  the  urine  a  few  drops  at  a  time  until 
the  last  trace  of  blue  just  disappears.  This  end-point  is 
easily  recognized.  During  the  whole  of  the  titration  the 
mixture  must  be  kept  vigorously  boiling.  Loss  by  evap- 
oration must  be  made  up  by  adding  water.  The  quantity 
of  urine  required  to  discharge  the  blue  color  contains  exactly 
0.05  gram  glucoSe,  and  the  percentage  contained  in  the 
original  sample  is  easily  calculated. 

(4)  Fermentation  Method.— This  is  convenient  and  satis- 
factory, its  chief  disadvantage  begin  the  time  required.  It  de- 
pends upon  the  fact  that  glucose  is  fermented  by  yeast  with 
evolution  of  CO2.  The  amount  of  gas  evolved  is  an  index  of 
the  amount  of  glucose.  Einhorn's  saccharimeter  (Fig.  29)  is 
the  simplest  apparatus. 

The  urine  must  be  so  diluted  as  to  contain  not  more  than 
I  per  cent,  of  glucose.  A  fragment  of  fresh  yeast  cake  about 
the  size  of  a  split-pea  is  mixed  with  a  definite  quantity  of  the 
urine  measured  in  the  tube  which  accompanies  the  appa- 
ratus. It  should  form  an  emulsion  free  from  lumps  or  air- 
bubbles.  The  long  arm  of  the  apparatus  is  then  filled  with 
the  mixture.  At  the  end  of  fifteen  to  twenty-four  hours  fer- 
mentation will  be  complete,  and  the  percentage  of  glucose  can 
be  read  off  upon  the  side  of  the  tube.  The  result  must  then 
be  multiplied  by  the  degree  of  dilution.  Since  yeast  itself 
sometimes  gives  ofif  gas,  a  control  test  must  be  carried  out 
with  normal  urine  and  the  amount  of  gas  evolved  must  be 
subtracted  from  that  of  the  test.  A  control  should  also  be 
made  with  a  known  glucose  solution  to  make  sure  that  the 
yeast  is  active. 

(5)  Robert's  Differential  Density  Method. — While  this 
method  gives  only  approximate  results,  it  is  convenient,  and 
requires  no  special  apparatus  but  an  accurate  urinometer. 
Mix  a  quarter  of  a  yeast-cake  with  about  4  oz.  of  urine. 
Take  the  specific  gravity  and  record  it.  Set  the  urine  in  a 
warm  place  for  twenty-four  hours  or  until  fermentation  is  com- 


CHEMIC  EXAMINATION  II 7 

plete.  Then  cool  to  the  temperature  at  which  the  specific 
gravity  was  originally  taken,  and  take  it  again.  The  differ- 
ence between  the  two  readings  gives  the  number  of  grains 
of  sugar  per  ounce,  and  this,  multiplied  by  0.234,  gives  the 
percentage  of  sugar.  If  the  original  reading  is  1.035,  ^^^  that 
after  fermentation  is  1.020,  the  urine  contains  1.035  — 1.020 
=  15  grains  of  sugar  per  fluidounce;  and  the  percentage  equals 
15X0.234  =  3.5. 

(2)  Levulose,  or  fruit-sugar,  is  very  rarely  present  in 
the  urine  except  in  association  with  glucose,  and  has 
about  the  same  significance.  Its  name  is  derived  from 
the  fact  that  it  rotates  polarized  light  to  the  left.  It  be- 
haves the  same  as  glucose  with  all  the  ordinary  tests, 
and  is  not  readily  distinguished  except  by  polarization. 

(3)  Lactose,  or  milk-sugar,  is  sometimes  present  in 
the- urine  of  nursing  women  and  in  that  of  women  who 
have  recently  miscarried.  It  is  of  interest  chiefly  be- 
cause it  may  be  mistaken  for  glucose.  //  reduces  copper, 
hut  does  not  ferment  with  yeast.  In  strong  solution  it  can 
form  crystals  with  phenylhydrazin,  but  is  extremely 
unlikely  to  do  so  when  the  test  is  applied  directly  to  the 
urine. 

(4)  Pentoses. — These  sugars  are  so  named  because 
they  contain  five  atoms  of  oxygen.  Vegetable  giuns 
form  their  chief  source.  They  reduce  copper  strongly 
but  slowly,  and  give  crystals  with  phenylhydrazin,  but 
do  not  ferment  with  yeast. 

Pentosuria  is  uncommon.  It  has  been  noted  after  in- 
gestion of  large  quantities  of  pentose-rich  substances, 
such  as  cherries,  plums,  and  fruit-juices,  and  is  said  to 
be  fairly  constant  in  habitual  use  of  morphin.  It  some- 
times accompanies  glycosuria  in  diabetes.     An  obscure 


Il8  THE   URINE 

chronic  form  of  pentosuria  without  cHnical  symptoms 
has  been  observed. 

Bial's  Orcin  Test. — Dextrose  is  first  removed  by  fermen- 
tation. About  5  c.c.  of  Bial's  reagent  are  heated  in  a  test- 
tube,  and  after  removing  from  the  flame  the  urine  is  added 
drop  by  drop,  not  exceeding  twenty  drops  in  all.  The  ap- 
pearance of  a  green  color  denotes  pentose. 

The  reagent  consists  of: 

30  i)er  cent,  hydrochloric  acid 500  c.c. 

10  ])er  cent,  ferric  chlorid  solution 25  drops 

Orcin i  gram. 

3.  Acetone  Bodies.-  -This  is  a  group  of  closely  related 
substances — acetone,  diacetic  acid,  and  beta-oxy butyric 
acid.  Acetone  is  derived  from  decomposition  of  diacetic 
acid,  and  this  in  turn  from  beta-oxybutyric  acid  by  oxida- 
tion. The  origin  of  beta-oxybutyric  acid  is  not  definitely 
known,  but  it  is  probable  that  its  chief,  if  not  its  only, 
source  is  in  some  obscure  metabolic  disturbance  with 
abnormal  destruction  of  fats.  The  three  substances 
generally  appear  in  the  urine  in  the  order  mentioned. 
When  the  disturbance  is  mild,  acetone  only  appears;  as  it 
becomes  more  marked,  diacetic  acid  is  added,  and  finally 
beta-oxybutyric  acid  appears.  The  {presence  of  beta- 
oxybutyric  acid  in  the  blood  is  probably  the  chief  cause 
of  the  form  of  auto-intoxication  known  as  "  acid  intoxi- 
cation." 

(i)  Acetone. — Minute  traces,  too  small  for  the  ordi- 
nary tests,  may  be  present  in  the  urine  under  normal 
conditions.  Larger  amounts  are  not  uncommon  in 
fevers,  gastro-intestinal  disturbances,  and  certain  ner- 
vous disorders.     A   notable   degree  of  acetonuria  has 


CHEMIC   EXAMINATION  II 9 

Kkewise  been  observed  in  pernicious  vomiting  of  preg- 
nancy and  in  eclampsia. 

Acetonuria  is  practically  always  observed  in  acid 
intoxication,  and,  together  with  diaceturia,  constitutes 
its  most  significant  diagnostic  sign.  A  similar  or  identi- 
cal toxic  condition,  always  accompanied  by  acetonuria 
and  often  fatal,  is  now  recognized  as  a  not  infrequent 
late  effect  of  anesthesia,  particularly  of  chloroform  anes- 
thesia. This  postanesthetic  toxemia  is  more  Hkely  to 
appear,  and  is  more  severe  when  the  urine  contains  any 
notable  amount  of  acetone  before  operation,  which  sug- 
gests the  importance  of  routine  examination  for  acetone 
in  surgical  cases. 

Acetone  is  present  in  considerable  amounts  in  many 
cases  of  diabetes  mellitus,  and  is  always  present  in  severe 
cases.  Its  amount  is  a  better  indication  of  the  severity 
of  the  disease  than  is  the  amount  of  sugar.  A  progres- 
sive increase  is  a  grave  prognostic  sign.  It  can  be 
diminished  temporarily  by  more  liberal  allowance  of 
carbohydrates  in  the  diet. 

According  to  Folin,  acetone  is  present  in  only  small 
amounts  in  these  conditions,  the  substance  shown  by 
the  usual  tests,  particularly  after  distillation  of  the 
urine,  being  really  diacetic  acid.  In  this  connection, 
Frommer's  test  is  to  be  recommended,  since  it  does  not 
require  distillation,  and  does  not  react  to  diacetic  acid 
unless  too  great  heat  is  applied. 

Detection  of  Acetone. — The  urine  may  be  tested  di- 
rectly, but  it  is  best  to  distil  it  after  adding  a  little  phos- 
phoric or  hydrochloric  acid  to  prevent  foaming,  and  to 
test  the  first  few  cubic  centimeters  of  distillate.  A 
simple  distilling  apparatus  is  shown  in  Fig.  30.     The 


I20 


THE    URINE 


test-tube  may  be  attached  to  the  delivery  tube  by  means 
of  a  two-hole  rubber  cork  as  shown,  the  second  hole 
serving  as  air  vent,  or,  what  is  much  less  satisfactory, 
it  may  be  tied  in  place  with  a  string.  Should  the  vapor 
not  condense  well,  the  test-tube  may  be  immersed  in  a 
glass  of  cold  water. 


I       " 


Fig.  30. — A  simple  distillins  apparatus. 


When  diacetic  acid  is  present,  a  considerable  pro- 
portion will  be  converted  into  acetone  during  distilla- 
tion. 


(i)  Giuining's  Test. — To  a  few  cubic  centimeters  of  urine 
or  distillate  in  a  test-tube  add  a  few  drops  of  tincture  of  iodin 
and  of  ammonia  alternately  until  a  heavy  black  cloud  appears. 
This  cloud  will  gradually  clear  up,  and  if  acetone  be  present, 
iodoform,  usually  crystalline,  will  separate  out.  The  iodoform 
can  be  recognized  by  its  odor,  especially  upon  heating  (there 


CHEMIC  EXAMINATION  121 

is  danger  of  explosion  if  the  mixture  be  heated  before  the 
black  cloud  disappears),  or  by  detection  of  the  crystals  mi- 
croscopically. The  latter  only  is  safe,  unless  one  has  an 
imusually  acute  sense  of  smell.  Iodoform  crystals  are  yel- 
lowish, six-pointed  stars  or  six-sided  plates  (Fig.  31), 

This  modification  of  Lieben's  test  is  less  sensitive  than  the 
original,  but  is  sufficient  for  all  clinical  work;  it  has  the  ad- 
vantage that  alcohol  does  not  cause  confusion,  and  especially 
that  the  sediment  of  iodoform  is  practically  always  crystalline. 
When  applied  directly  to  the  urine,  phosphates  are  precipi- 


.  ^^tCs^T — - 

L-.     ., 

Fig.  31. — Iodoform  crystals  obtained  in  several  tests  for  acetone  by  Gunning's  method 
(X  about  600). 

tated  and  may  form  star-shaped  crystals  which  are  very  con- 
fusing to  the  inexperienced.  Albumin  prevents  formation 
of  the  crystals,  and  when  it  is  present,  the  urine  must  be  dis- 
tilled for  the  test. 

(2)  Lange's  Test. — This  is  a  modification  of  the  well- 
known  Legal  test.  It  is  more  sensitive  and  gives  a  sharper 
end-reaction.  To  a  small  quantity  of  urine  add  about  one- 
twentieth  its  volume  (i  drop  for  each  i  c.c.)  of  glacial  acetic 
acid  and  a  few  drops  of  fresh  concentrated  aqueous  solution  of 
sodium  nitroprussid,  and  gently  run  a  little  ammonia  upon  its 


122  THE    URINE 

surface.  If  acetone  be  present,  a  purple  ring  will  form  within 
a  few  minutes  at  the  junction  of  the  two  fluids. 

(3)  Frommer's  Test. — This  test  has  proved  very  satis- 
factory in  the  hands  of  the  writer.  The  urine  need  not  be 
distilled.  Alkalinize  about  10  c.c.  of  the  urine  with  2  or  3  c.c. 
of  40  per  cent,  caustic  soda  solution,  add  10  or  12  drops  of 
10  per  cent,  alcoholic  solution  of  salicylous  acid  (salicyl 
aldehyd),  heat  the  upper  portion  to  about  70°  C.  (it  should 
not  reach  the  boiling-point),  and  keep  at  this  temperature 
five  minutes  or  longer.  In  the  presence  of  acetone  an  orange 
color,  changing  to  deep  red,  appears  in  the  heated  portion. 

The  test  can  be  made  more  definite  by  adding  the  caustic 
soda  in  substance  (about  i  gram),  and  before  it  goes  into 
solution  adding  the  salicyl  aldehyd  and  warming  the  lower 
portion. 

(2)  Diacetic  acid  occurs  in  the  same  conditions  as 
acetone,  but  is  less  frequent  and  has  more  serious  signifi- 
cance. In  diabetes  its  presence  is  a  grave  symptom  and 
often  forewarns  of  approaching  coma.  It  rarely  or  never 
occurs  without  acetone. 

Detection: — The  urine  must  be  fresh. 

(i)  Gerhardt's  Test. — To  a  few  cubic  centimeters  of  the 
urine  add  solution  of  ferric  chlorid  (about  10  per  cent.)  drop 
by  drop  until  the  phosphates  are  precipitated;  filter  and  add 
more  of  the  ferric  chlorid.  If  diacetic  acid  be  present,  the 
urine  will  assume  a  Bordeaux-red  color  which  disappears 
upon  boiling.  A  red  or  violet  color  which  does  not  disappear 
upon  boiling  may  be  produced  by  other  substances,  as  phenol, 
salicylates,  and  antipyrin. 

(2)  Lindemann's  Test. — To  about  10  c.c.  of  urine  add 
5  drops  30  per  cent,  acetic  acid,  5  drops  Lugol's  solution,  and 
2  or  3  c.c.  chloroform,  and  shake.  The  chloroform  does  not 
change  color  if  diacetic  acid  be  present,  but  becomes  reddish 


CHEMIC  EXAMINATION  1 23 

violet  in  its  absence.    This  test  is  claimed  by  its  advocates  to 
be  more  sensitive  and  more  reliable  than  Gerhardt's. 

(3)  Oxybutyric  acid  has  much  the  same  significance 
as  diacetic  acid,  but  is  of  more  serious  import.  There  is 
no  satisfactory  clinical  test  for  it. 

4.  Bile. — Bile  appears  in  the  urine  in  all  diseases  which 
produce  jaundice,  often  some  days  before  the  skin  be- 
comes yellow;  and  in  many  disorders  of  the  liver  not 
severe  enough  to  cause  jaundice.  It  also  occurs  in  dis- 
eases with  extensive  and  rapid  destruction  of  red  blood- 
corpuscles.  Both  bile-pigment  and  bile  acids  may  be 
found.  They  generally  occur  together,  but  the  pigment 
is  not  infrequently  present  alone.  Bilirubin,  only,  oc- 
curs in  freshly  voided  urine,  the  other  pigments  (bili- 
verdin,  bilifuscin,  etc.)  being  produced  from  this  by 
oxidation  as  the  urine  stands.  The  acids  are  almost 
never  present  without  the  pigments,  and  are,  therefore, 
seldom  tested  for  clinically. 

Detection  of  Bile-pigment. — Bile-pigment  gives  the 
urine  a  greenish-yellow,  yellow,  or  brown  color,  which 
upon  shaking  is  imparted  to  the  foam.  Cells,  casts,  and 
other  structures  in  the  sediment  may  be  stained  brown  or 
yellow.  This,  however,  should  not  be  accepted  as  prov- 
ing the  presence  of  bile  without  further  tests. 

(i)  Smith's  Test. — Overlay  the  urine  with  tincture  of  iodin 
diluted  with  nine  times  its  volume  of  alcohol.  An  emerald- 
green  ring  at  the  zone  of  contact  shows  the  presence  of  bile- 
pigments.  It  is  convenient  to  use  a  conical  test-glass,  one 
side  of  which  is  painted  white. 

(2)  Gmelin's  Test. — This  consists  in  bringing  shghtly 
yellow  nitric  acid  into  contact  with  the  urine.     A  play  of 


124  THE    URINE 

colors,  of  which  green  and  violet  are  most  distinctive,  denotes 
the  presence  of  bile-pigment.  Colorless  nitric  acid  will  be- 
come yellow  upon  standing  in  the  sunlight.  The  test  may  be 
applied  in  various  ways :  by  overlaying  the  acid  with  the  urine; 
by  bringing  a  drop  of  each  together  upon  a  porcelain  plate; 
by  filtering  the  urine  through  thick  filter-paper,  and  touching 
the  paper  with  a  drop  of  the  acid;  and,  probably  best  of  all, 
by  precipitating  with  lime-water,  filtering,  and  touching  the 
precipitate  with  a  drop  of  the  acid.  In  the  last  method  bili- 
rubin is  carried  down  as  an  insoluble  calcium  compound. 

Detection  of  Bile  Acids. — Hay's  test  is  simple,  sensi- 
tive, and  fairly  reliable,  and  will,  therefore,  appeal  to 
the  practitioner.  It  depends  upon  the  fact  that  bile 
acids  lower  surface  tension.  Other  tests  require  isola- 
tion of  the  acids  for  any  degree  of  accuracy. 

Hay's  Test. — Upon  the  surface  of  the  urine,  which  must  not 
be  warm,  sprinkle  a  little  finely  powdered  sulphur.  If  it 
sinks  at  once,  bile  acids  are  present  to  the  amount  of  o.oi 
per  cent,  or  more;  if  only  after  gentle  shaking,  0.0025  P^r 
cent,  or  more.  If  it  remains  floating,  even  after  gentle 
shaking,  bile  acids  are  absent. 

5.  Hemoglobin. — The  presence  in  the  urine  of  hemo- 
globin or  pigments  directly  derived  from  it,  accompanied 
by  few,  if  any,  red  corpuscles,  constitutes  hemoglobinuria. 
It  is  a  rare  condition,  and  must  be  distinguished  from 
hematuria,  or  Uood  in  the  urine,  which  is  common.  In 
both  conditions  chemic  tests  will  show  hemoglobin,  but 
in  the  latter  the  microscope  will  reveal  the  presence  of 
red  corpuscles.  Urines  which  contain  notable  amounts 
of  hemoglobin  have  a  reddish  or  brown  color,  and  may 
deposit  a  sediment  of  brown,  granular  pigment. 


CHEMIC  EXAMINATION  I25 

Hemoglobinuria  occurs  when  there  is  such  extensive 
destruction  of  red  blood-cells  within  the  body  that  the 
liver  cannot  transform  all  the  hemoglobin  set  free  into 
bile-pigment.  The  most  important  examples  are  seen  in 
poisoning,  as  by  mushrooms  and  potassimn  chlorate, 
in  scurvy  and  purpura,  in  malignant  malaria  (black water 
.fever),  and  in  the  obscure  condition  known  as  "  paroxys- 
mal hemoglobinuria."  This  last  is  characterized  by  the 
appearance  of  large  quantities  of  hemoglobin  at  inter- 
vals, usually  following  exposure  to  cold,  the  urine  remain- 
ing free  from  hemoglobin  between  the  attacks. 

Detection. — Teichmann's  test  (p.  274)  may  be  applied 
to  the  precipitate  after  boiling  and  filtering,  but  the 
guaiac  test  is  more  convenient  in  routine  work. 

Guaiac  Test. — Mix  equal  parts  of  "  ozonized  "  turpentine 
and  fresh  tincture  of  guaiac  which  has  been  diluted  with 
alcohol  to  a  light  sherry- wine  color.  In  a  test-tube  or  conical 
glass  overlay  the  urine  with  this  mixture.  A  bright  blue  ring 
will  appear  at  the  zone  of  contact  within  a  few  minutes  if 
hemoglobin  be  present.  The  guaiac  should  be  kept  in  an 
amber-colored  bottle.  Fresh  turpentine  can  be  "  ozonized  " 
by  allowing  it  to  stand  a  few  days  in  an  open  vessel  in  the 
sunlight. 

This  test  is  very  sensitive,  and  a  negative  result  proves  the 
absence  of  hemoglobin.  Positive. results  are  not  conclusive, 
because  numerous  other  substances — few  of  them  likely  to  be 
found  in  the  urine — may  produce  the  blue  color.  That  most 
likely  to  cause  confusion  is  pus,  but  the  blue  color  produced 
by  it  disappears  upon  heating.  The  thin  film  of  copper 
often  left  in  a  test-tube  after  testing  for  sugar  may  give  the 
reaction,  as  may  also  the  fumes  from  an  open  bottle  of  bromin. 


126  THE    URINE 

6.  Alkapton  Bodies.- -The  name,  alkaptonuria,  has 
been  given  to  a  condition  in  which  the  urine  turns 
reddish-brown  upon  standing  and  strongly  reduces 
copper  (but  not  bismuth),  owing  to  the  presence  of 
certain  substances  which  result  from  imperfect  protein 
metabolism.  The  change  of  color  takes  place  quickly 
when  fresh  urine  is  alkalinized,  hence  the  name,  alkapton 
bodies. 

Alkaptonuria  is  unaccompanied  by  other  symptoms, 
and  has  little  clinical  importance.  Only  about  forty-five 
cases,  mostly  congenital,  have  been  reported.  The 
change  in  color  of  the  urine  and  the  reduction  of  copper 
with  no  reduction  of  bismuth  nor  fermentation  with 
yeast  would  suggest  the  condition. 

7.  Melanin. — Urine  which  contains  melanin  likewise 
darkens  upon  exposure  to  the  air,  assuming  a  dark 
brown  or  black  color.  This  is  due  to  the  fact  that  the 
substance  is  eliminated  as  a  chromogen — melanogen — 
which  is  later  converted  into  the  pigment. 

Melanuria  occurs  in  most,  but  not  all,  cases  of  mela- 
notic sarcoma.  Its  diagnostic  value  is  lessened  by  the 
fact  that  it  has  been  observed  in  other  wasting  diseases. 

Tests  for  Melanin. — (i)  Addition  of  ferric  chlorid  gives  a 
gray  precipitate  which  blackens  on  standing. 

(2)  Bromin  water  causes  a  yellow  precipitate  which 
gradually  turns  black. 

8.  Diazo  Substances. — Certain  unknown  substances 
sonretimes  present  in  the  urine  give  a  characteristic 
color  reaction — the  "  diazo  reaction  "  of  Ehrlich — when 
treated  with  diazo-benzol-sulphonic  acid  and  ammonia. 


CHEMIC  EXAMINATION  1 27 

This  reaction  has  much  cUnical  value,  provided  its  limi- 
tations be  recognized.  It  is  at  best  an  empirical  test 
and  must  be  interpreted  in  the  light  of  cHnical  symptoms. 
Although  it  has  been  met  with  in  a  considerable  number 
of  diseases,  its  usefulness  is  practically  limited  to  ty- 
phoid fever,  tuberculosis,  and  measles. 

(i)  Tjrphoid  Fever. — Practically  all  cases  give  a 
positive  reaction,  which  varies  in  intensity  with  the 
severity  of  the  disease.  It  is  so  constantly  present  that 
it  is  sometimes  said  to  be  "  negatively  pathognomonic  ": 
if  negative  upon  several  successive  days  at  a  stage  of  the 
disease  when  it  should  be  positive,  typhoid  is  almost 
certainly  absent.  Upon  the  other  hand,  a  reaction 
when  the  urine  is  highly  diluted  (i :  50  or  more)  has 
much  positive  diagnostic  value,  since  this  dilution  pre- 
vents the  reaction  in  most  conditions  which  might  be 
mistaken  for  typhoid;  but  it  should  be  noted  that  mild 
cases  of  typhoid  may  not  give  it  at  this  dilution.  Ordi- 
narily the  diazo  appears  a  little  earlier  than  the  Widal 
reaction, — about  the  fourth  or  fifth  day, — but  it  may  be 
delayed.  In  contrast  to  the  Widal,  it  begins  to  fade 
about  the  end  of  the  second  week,  and  soon  thereafter 
entirely  disappears.  An  early  disappearance  is  a  favor- 
able sign.  It  reappears  during  a  relapse,  and  thus  helps 
to  distinguish  between  a  relapse  and  a  complication,  in 
which  it  does  not  reappear. 

(2)  Tuberculosis. — The  diazo  reaction  has  been  ob- 
tained in  many  forms  of  the  disease.  It  has  little  or 
no  diagnostic  value.  Its  continued  presence  in  pul- 
monary tuberculosis  is,  however,  a  grave  prognostic 
sign,  even  when  the  physical  signs  are  slight.  After  it 
once  appears  it  generally  persists  more  or  less  intermit- 


128  THE    URINE 

tently  until  death,  the  average  length  of  life  after  its 
appearance  being  about  six  months.  The  reaction  is 
often  temporarily  present  in  mild  cases  during  febrile 
complications,  and  has  then  no  significance. 

(3)  Measles. — A  positive  reaction  is  usually  obtained 
in  measles,  and  may  help  to  distinguish  this  disease 
from  German  measles,  in  which  it  does  not  occur.  It 
generally  appears  before  the  eruption  and  remains  about 
five  days. 

Technic. — Although  the  test  is  really  a  very  simple  one, 
careful  attention  to  technic  is  imperative.  Many  of  the  early 
workers  were  very  lax  in  this  regard.  Faulty  technic  and 
failure  to  record  the  stage  of  the  disease  in  which  the  tests 
were  made  have  probably  been  responsible  for  the  bulk  of  the 
conflicting  results  reported. 

Certain  drugs  often  given  in  tuberculosis  and  typhoid 
interfere  with  or  prevent  the  reaction.  The  chief  are  creosote, 
tannic  acid  and  its  compounds,  opium  and  its  alkaloids,  salol, 
phenol,  and  the  iodids.     The  reagents  are: 

(i)  Saturated  solution  sulphanilic  acid  in  5  per  cent, 
hydrochloric  acid. 

(2)  0.5  per  cent,  aqueous  solution  sodium  nitrite. 

(3)  Strong  ammonia. 

Mix  100  parts  of  (i)  and  one  part  of  (2).  In  a  test-tube 
take  equal  parts  of  this  mixture  and  the  urine,  and  pour  i  or 
2  c.c.  of  the  ammonia  upon  its  surface.  If  the  reaction  be 
positive,  a  garnet  ring  will  form  at  the  junction  of  the  two 
fluids;  and  upon  shaking,  a  distinct  pink  color  will  be  imparted 
to  the  foam.  The  color  of  the  foam  is  the  essential  feature. 
If  desired,  the  mixture  may  be  well  shaken  before  the  ammonia 
is  added:  the  pink  color  will  then  instantly  appear  in  that 
portion  of  the  foam  which  the  ammonia  has  reached,  and  can 
be  readily  seen.    The  color  varies  from  eosin-pink  to  deep 


CHEMIC  EXAMINATION  1 29 

crimson,  depending  upon  the  intensity  of  the  reaction.  It  is 
a  pure  pink  or  red;  any  trace  of  yellow  or  orange  denotes  a 
negative  reaction.  A  doubtful  reaction  should  be  considered 
negative. 

9.  Pancreatic  Reaction.— Cammidge  has  shown  that 
in  cases  of  pancreatitis  a  substance  capable  of  forming 
crystals  with  phenylhydrazin  can  be  developed  by  boiling 
the  urine  with  a  mineral  acid,  and  has  offered  the  follow- 
ing test  as  an  aid  in  diagnosis  of  this  obscure  condition. 
The  nature  both  of  this  substance  and  the  antecedent 
substance  from  which  it  is  derived  is  not  known.  As 
originally  proposed,  the  test  was  complicated  and  prob- 
ably not  trustworthy,  but  with  his  improved  and  sim- 
plified technic,  Cammidge  has  had  very  promising  results. 
In  200  consecutive  examinations  in  which  the  diagnosis 
was  confirmed,  postmortem  or  at  operation,  67  cases  of 
pancreatitis  (65  chronic,  2  acute)  gave  positive  reactions; 
4  cases  of  cancer  of  the  pancreas  were  positive,  1 2  nega- 
tive; 4  cases  in  which  no  pancreatitis  was  found  were 
positive,  113  were  negative.  Normal  urines  do  not  give 
the  reaction.  The  difficulty  and  importance  of  diag- 
nosis in  pancreatitis  warrant  inclusion  of  the  method 
here,  even  though  more  recent  work  indicates  that  its 
value  is  not  so  great  as  originally  claimed. 

While  the  test  is  somewhat  tedious,  all  the  manipula- 
tions are  simple  and  require  no  apparatus  but  flasks, 
test-tubes,  and  funnels. 

Technic. — Careful  attention  to  detail  is  imperative.    An 

ordinary  routine  examination  is  first  made.     Albumin  and 

sugar,  if  present,  must  be  removed:  the  former,  by  acidifying 

with  acetic  acid,  boiling,  and  filtering;  the  latter,  by  fermenta- 

9 


I30 


THE    URINE 


tion  with  yeast  after  the  first  step  of  the  method  proper.  An 
alkahne  urine  should  be  made  sUghtly  acid  with  hydrochloric 
acid. 

(i)  Forty  cubic  centimeters  of  the  urine,  which  has  been 
rendered  perfectly  clear  by  repeated  filtration  through  the 
same  filter-paper  are  placed  in  a  small  flask,  treated  with  i 
c.c.  concentrated  hydrochloric  acid  and  gently  boiled  on  a 


Fig.  32. — "Pancreatic  rcacti^ 


1"   ll,lsk■^  tilti'il   with   fuiiiu'l  conilcnsers  on  a  sand-bath 
(Rohson  and  Cammidge). 


sand-bath  for  ten  minutes,  a  funnel  wath  long  stem  being 
placed  in  the  neck  of  the  flask  to  act  as  a  condenser  (Fig.  32). 
After  boiling,  the  urine  is  cooled  in  a  stream  of  cold  water  and 
brought  to  its  original  bulk  with  distilled  w^ater;  8  gm.  of 
lead  carbonate  are  then  added  to  neutralize  the  acid.  The 
fluid  is  allowed  to  stand  a  few  minutes  and  then  filtered 
through  well-moistened  fine-grain  filter-paper  until  perfectly 
clear. 


CHEMIC  EXAMINATION  131 

(2)  The  filtrate  is  shaken  up  with  8  gm.  powdered  tribasic 
lead  acetate  and  filtered.  The  excess  of  lead  is  then  removed 
by  passing  hydrogen  sulphid  gas  through  the  fluid  (see  page 
135)  or  by  shaking  well  with  4  gm.  finely  powdered  sodium 
sulphate,  heating  to  boiling,  cooling  to  as  low  a  temperature 
as  possible  in  a  stream  of  water,  and  filtering  as  before  until 
perfectly  clear. 


Fig.  33. — Improved  "pancreatic  reaction."  Crystals  obtained  from  a  case  of  chronic 
pancreatitis  with  gall-stones  in  the  common  duct  (X200)  (from  a  photo  by  P.  J.  Cam- 
midge). 

(3)  Ten  cubic  centimeters  of  the  filtrate  are  then  made 
up  to  17  c.c.  with  distilled  water,  and  added  to  a  mixture  of 
0.8  gm.  phenylhydrazin  hydrochlorate,  2  gm.  powdered  so- 
dium acetate,  and  i  c.c.  50  per  cent,  acetic  acid  in  a  small  flask 
with  funnel  condenser.  This  is  boiled  on  a  sand-bath  for  ten 
minutes,  and  filtered  while  hot  through  filter-paper  moistened 
with  hot  water  into  a  test-tube  with  a  15  c.c.  mark.     Should 


132 


THE    URINE 


the  filtrate  not  reach  this  mark,  make  up  to  15  c.c.  with  hot 
distilled  water.     Allow  to  cool  slowly. 

(4)  In  well-marked  cases  of  pancreatitis  a  yellow  pre- 
cipitate appears  within  a  few  hours;  in  milder  cases,  it  may  not 
appear  for  twelve  hours.  The  microscope  shows  this  sediment 
to  consist  of  "  Ions;;,  light  yellow,  flexible,  hair-like  crystals 
arranged  in  sheaves,  which,  when  irrigated  with  33  per  cent. 
suljihuric  acid,  melt  away  and  disappear  in  ten  to  fifteen  sec- 
onds after  the  acid  first  touches  them  "  (Fig.  33). 

(5)  To  exclude  traces  of  glucose  which  might  be  overlooked 
in  the  preliminary  examination  a  control  test  should  be  carried 
out  in  the  same  manner  with  omission  of  step  i. 

10.  Drugs.—  The  effect  of  various  drugs  upon  the 
color  of  the  urine  has  been  mentioned  (p.  71).  Most 
poisons  are  eliminated  in  the  urine,  but  their  detection 
is  more  properly  discussed  in  works  upon  toxicology.  A 
few  drugs  which  are  of  interest  to  the  practitioner,  and 
which  can  be  detected  by  comparatively  simple  methods, 
are  mentioned  here. 

Acetanilid  and  Phenacetin. — The  urine  is  evaporated 
by  gentle  heat  to  about  half  its  volume,  boiled  for  a  few 
minutes  with  about  one-fifth  its  volume  of  strong  hydro- 
chloric acid,  and  shaken  out  with  ether.  The  ether  is 
evaporated,  the  residue  dissolved  in  water,  and  the 
following  test  applied :  To  about  10  c.c.  are  added  a  few 
cubic  centimeters  of  3  per  cent,  phenol,  followed  by  a 
weak  solution  of  chromium  trioxid  (chromic  acid)  drop 
by  drop.  The  fluid  assumes  a  red  color,  which  changes 
to  blue  when  ammonia  is  added.  If  the  urine  is  very 
pale,  extraction  with  ether  may  be  omitted. 

Antipjn-in. — This  drug  gives  a  dark-red  color  when  a 
few  drops  of  10  per  cent,  ferric  chlorid  are  added  to  the 


CHEMIC  EXAMINATION  133 

urine.  The  color  does  not  disappear  upon  boiling,  which 
excludes  diacetic  acid. 

Arsenic. — Reinsch's  Test. — Add  to  the  urine  in  a  test- 
tube  or  small  flask  about  one-seventh  its  volume  of  hy- 
drochloric acid,  introduce  a  piece  of  bright  copper-foil 
about  one-eighth  inch  square,  and  boil  for  several  min- 
utes. If  arsenic  be  present,  a  dark-gray  film  is  deposited 
upon  the  copper.  The  test  is  more  delicate  if  the  urine 
be  concentrated  by  slow  evaporation.  This  test  is  well 
known  and  is  widely  used,  but  is  not  so  reUable  as  the 
following. 

GutzeWs  Test. — In  a  large  test-tube  place  a  little 
arsenic-free  zinc,  and  add  5  to  10  c.c.  pure  dilute  hydro- 
chloric acid  and  a  few  drops  of  iodin  solution  (Gram's 
solution  will  answer),  then  add  5  to  10  c.c.  of  the  urine. 
At  once  cover  the  mouth  of  the  tube  with  a  filter-paper 
cap  moistened  with  saturated  aqueous  solution  of  silver 
nitrate  (i:  i).  If  arsenic  be  present,  the  paper  quickly 
becomes  lemon-yellow,  owing  to  formation  of  a  com- 
pound of  silver  arsenid  and  silver  nitrate,  and  turns  black 
when  touched  with  a  drop  of  water.  To  make  sure  that 
the  reagents  are  arsenic-free,  the  paper  cap  may  be  ap- 
plied for  a  few  minutes  before  the  urine  is  added. 

Atropin  will  cause  dilatation  of  the  pupil  when  a  few 
drops  of  the  urine  are  placed  in  the  eye  of  a  cat  or  rabbit. 

Bromids  can  be  detected  by  acidifying  about  10  c.c.  of 
the  urine  with  dilute  sulphuric  acid,  adding  a  few  drops 
of  fuming  nitric  acid  and  a  few  cubic  centimeters  of 
chloroform,  and  shaking.  In  the  presence  of  bromin  the 
chloroform,  which  settles  to  the  bottom,  assumes  a  yellow 
color. 

Iodin  from   ingestion   of   iodids   or   absorption  from 


134  THE   URINE 

iodoform  dressings  is  tested  for  in  the  same  way  as  the 
bromids,  the  chloroform  assuming  a  pink  to  reddish- 
violet  color.  To  detect  traces,  a  large  quantity  of  urine 
should  be  rendered  alkaline  with  sodium  carbonate  and 
great Iv  concentrated  by  evaporation  before  testing. 

Lead.  No  simple  method  is  sufficiently  sensitive  to 
detect  the  traces  of  lead  which  occur  in  the  urine  in 
chronic  poisoning.  Of  the  more  sensitive  methods,  that 
of  Arthur  Lederer  is  probably  best  suited  to  the  prac- 
titioner: 

It  is  essential  that  all  apparatus  used  be  lead-free. 
Five  hundred  cubic  centimeters  of  the  urine  are  acidified 
with  70  c.c.  pure  sulphuric  acid,  and  heated  in  a  beaker 
or  porcelain  dish.  About  20  to  25  gm.  of  potassium 
persulphate  are  added  a  little  at  a  time.  This  should 
decolorize  the  urine,  leaving  it  only  slightly  yellow.  If 
it  darkens  upon  heating,  a  few  more  crystals  of  potassium 
persulphate  are  added,  the  burner  being  first  removed  to 
prevent  boiling  over ;  if  it  becomes  cloudy,  a  small  amount 
of  sulphuric  acid  is  added.  It  is  then  boiled  until  it  has 
evaporated  to  250  c.c.  or  less.  After  cooling,  an  equal 
volume  of  alcohol  is  added,  and  the  mixture  allowed  to 
stand  in  a  cool  place  for  four  or  five  hours,  during  which 
time  all  the  lead  will  be  precipitated  as  insoluble  sulphate. 

The  mixture  is  then  filtered  through  a  small,  close- 
grained  filter-paper  (preferably  an  ashless,  quantitative 
filter-paper),  and  any  sediment  remaining  in  the  beaker 
or  dish  is  carefully  washed  out  with  alcohol  and  filtered. 
A  test-tube  is  placed  underneath  the  funnel;  a  hole  is 
punched  through  the  tip  of_the  filter  with  a  small  glass  rod, 
and  all  the  precipitate  (which  may  be  so  slight  as  to  be 
scarcely  visible)  washed  down  into  the  test-tube  with  a 


CHEMIC  EXAMINATION  135 

jet  of  distilled  water  from  a  wash-bottle,  using  as  little 
water  as  possible.  Ten  cubic  centimeters  will  usually 
sufl&ce.  This  fluid  is  then  heated,  adding  crystals  of 
sodium  acetate  until  it  becomes  perfectly  clear.  It  now 
contains  all  the  lead  of  the  5cxd  c.c.  urine  in  the  form  of 
lead  acetate.  It  is  allowed  to  cool,  and  hydrogen  sulphid 
gas  is  passed  through  it  for  about  five  minutes.  The 
slightest  yellowish-brown  discoloration  indicates  the  pres- 
ence of  lead.    A  very  slight  discoloration  can  be  best  seen 


Fig.  34. — A  simple  hydrogen  sulphid  generator. 

when  looked  at  from  above.  For  comparison,  the  gas 
may  be  passed  through  a  test-tube  containing  an  equal 
amount  of  distilled  water.  The  quantity  of  lead  can  be 
determined  by  comparing  the  discoloration  with  that 
produced  by  passing  the  gas  through  lead  acetate  (sugar 
of  lead)  solutions  of  known  strength.  One  gram  of  lead 
acetate  crystals  contains  0.54  gram  of  lead.  Hydrogen 
sulphid  is  easily  prepared  in  the  simple  apparatus  shown 
in  Fig.  34.     A  small  quantity  of  iron  sulphid  is  placed 


136  THE   URINE 

in  the  test-tube;  a  little  dilute  hydrochloric  acid  is  added; 
the  cork  is  replaced;  and  the  delivery  tube  is  inserted  to 
the  bottom  of  the  fluid  to  be  tested. 

Mercury. — Traces  can  be  detected  in  the  urine  for  a 
considerable  time  after  the  use  of  mercury  compounds 
by  ingestion  or  inunction. 

About  a  liter  of  urine  is  acidified  with  10  c.c.  hydro- 
chloric acid,  and  a  small  piece  of  copper-foil  or  gauze  is 
introduced.  This  is  gently  heated  for  an  hour,  and 
allowed  to  stand  for  twenty-four  hours.  The  metal  is 
then  removed,  and  washed  successively  with  very  dilute 
sodium  hydroxid  solution,  alcohol,  and  ether.  When 
dry.  it  is  placed  in  a  long,  slender  test-tube,  and  the  lower 
portion  of  the  tube  is  heated  to  redness.  If  mercury  be 
present,  it  will  volatilize  and  condense  in  the  upper  por- 
tion of  the  tube  as  small,  shining  globules  which  can  be 
seen  with  a  hand-magnifier  or  low  power  of  the  micro- 
scope. If,  now,  a  crystal  of  iodin  be  dropped  into  the 
tube  and  gently  heated,  the  mercury  upon  the  side  of  the 
tube  is  changed  first  to  the  yellow  iodid,  and  later  to  the 
red  iodid,  which  are  recognized  by  their  color. 

Morphin. — Add  sufficient  ammonia  to  the  urine  to 
render  it  distinctly  ammoniacal,  and  shake  thoroughly 
with  a  considerable  quantity  of  pure  acetic  ether.  Sepa- 
rate the  ether  and  evaporate  to  dryness.  To  a  httle  of 
the  residue  in  a  watch-glass  or  porcelain  dish  add  a  few 
drops  of  formaldehyd-sulphuric  acid,  which  has  been 
freshly  prepared  by  adding  one  drop  of  formalin  to  i  c.c. 
pure  concentrated  sulphuric  acid.  If  morphin  be  pres- 
ent, this  will  produce  a  purple-red  color,  which  changes 
to  violet,  blue-violet,  and  finally  nearly  pure  blue. 

Phenol. — As   has   been   stated,    the   urine   following 


CHEMIC  EXAMINATION  137 

phenol  poisoning  turns  olive-green  and  then  brownish- 
black  upon  standing.  Tests  are  of  value  in  recognizing 
poisoning  from  ingestion  and  in  detecting  absorption 
from  carbolized  dressings. 

The  urine  is  acidulated  with  hydrochloric  acid  and 
distilled.  To  the  first  few  cubic  centimeters  of  distillate 
is  added  lo  per  cent,  solution  of  ferric  chlorid  drop  by 
drop.  The  presence  of  phenol  causes  a  deep  amethyst- 
blue  color,  as  in  Uffelmann's  test  for  lactic  acid. 

Phenolphthalein,  which  is  now  widely  used  as  a  ca- 
thartic, gives  a  bright  pink  color  when  the  urine  is  ren- 
dered alkaline  with  caustic  soda. 

Quinin. — A  considerable  quantity  of  the  urine  is  ren- 
dered alkaline  with  ammonia  and  extracted  with  ether; 
the  ether  is  evaporated,  and  a  portion  of  the  residue  dis- 
solved in  about  twenty  drops  of  dilute  alcohol.  The 
alcoholic  solution  is  acidulated  with  dilute  sulphuric 
acid,  a  drop  of  an  alcoholic  solution  of  iodin  (tincture 
of  iodin  diluted  about  ten  times)  is  added,  and  the  mix- 
ture, is  warmed.  Upon  cooling,  an  iodin  compound  of 
quinin  (herapathite)  will  separate  out  in  the  form  of  a 
microcrystalline  sediment  of  green  plates. 

The  remainder  of  the  residue  may  be  dissolved  in  a 
little  dilute  sulphuric  acid.  This  solution  will  show  a 
characteristic  blue  fluorescence  when  quinin  is  present. 

Resinous  drugs  cause  a  white  precipitate  like  that  of 
albumin  when  strong  nitric  acid  is  added  to  the  urine. 
This  is  dissolved  by  alcohol. 

Salicylates,  salol,  and  similar  drugs  give  a  bluish- 
violet  color,  which  disappears  upon  heating,  upon  addi- 
tion of  a  few  drops  of  10  per  cent,  ferric  chlorid  solution. 
When  the  quantity  of  salicylates  is  small,  the  urine  may 


138  THE    URINE 

be  acidified  with  hydrochloric  acid  and  extracted  with 
ether,  the  ether  evaporated,  and  the  test  applied  to  an 
aqueous  solution  of  the  residue. 

Tannin  and  its  compounds  appear  in  the  urine  as 
gallic  acid,  and  the  urine  becomes  greenish-black  (inky, 
if  much  gallic  acid  be  present)  when  treated  with  a  solu- 
tion of  ferric  chlorid. 

III.  MICROSCOPIC  EXAMINATION 

A  careful  microscopic  examination  will  often  reveal 
structures  of  great  diagnostic  importance  in  urine  which 
seems  perfectly  clear,  and  from  which  only  very  slight 
sediment  can  be  obtained  with  the  centrifuge.  Upon  the 
other  hand,  cloudy  urines  with  abundant  sediment  are 
often  shown  by  the  microscope  to  contain  nothing  of 
clinical  significance. 

Since  the  nature  of  the  sediment  soon  changes,  the 
urine  must  be  examined  while  fresh,  preferably  within  six 
hours  after  it  is  voided.  The  sediment  is  best  obtained 
by  means  of  the  centrifuge.  If  a  centrifuge  is  not 
available,  the  urine  may  be  allowed  to  stand  in  a  conical 
test-glass  for  six  to  twenty-four  hours  after  adding  some 
preservative  (p.  69).  The  "  torfuge  "  (Fig.  35)  is  said 
to  be  a  very  satisfactory  substitute  for  the  centrifuge, 
and  is  readily  portable. 

A  small  amount  of  the  sediment  should  be  transferred 
to  a  slide  by  means  of  a  pipet.  It  is  very  important  to  do 
this  properly.  The  best  pipet  is  a  small  glass  tube  which 
has  been  drawn  out  at  one  end  to  a  tip  with  rather  small 
opening.  The  tube  or  glass  containing  the  sediment  is 
held  on  a  level  with  the  eye.  the  larger  end  of  the  pipet  is 
closed  with  the  index-finger,  which  must  be  dry,  and  the 


MICROSCOPIC  EXAMINATION 


139 


tip  is  carried  down  into  the  sediment.  By  carefully 
loosening  the  finger,  but  not  entirely  removing  it,  a  small 
amount  of  the  sediment  is  then  allowed  to  run  slowly  into 
the  pipet.  Slightly  rotating  the  pipet  will  aid  in  accom- 
plishing this,  and  at  the  same  time  will  serve  to  loosen 
any  structures  which  cling  to  the  bottom  of  the  tube. 
After  wiping  off  the  urine  which  adheres  to  the  outside, 
a  drop  from  the  pipet  is  placed  upon  a  clean  slide. 
A  hair  is  then  placed  in  the  drop,  and  a  large  cover-glass 


Fig.  35. — Wetherill's  torfuge. 

applied.  Many  workers  use  no  cover.  This  offers  a 
thicker  layer  and  larger  area  of  urine,  the  chance  of  find- 
ing scanty  structures  being  proportionately  increased.  It 
has  the  disadvantage  that  any  jarring  of  the  room  (as  by 
persons  walking  about)  sets  the  microscopic  field  into 
vibratory  motion  and  makes  it  impossible  to  see  an3^thing 
clearly;  and  since  it  does  not  allow  of  the  use  of  high- 
power  objectives,  one  cannot  examine  details  as  one  often 
wishes  to  do.  A  large  cover-glass  with  a  hair  beneath  it 
avoids  these  disadvantages,  and  gives  enough  urine  to 
find    any    structures   which    are    present   in    sufficient 


I40  THE   URINE 

number  to  have  clinical  significance,  provided  other 
points  in  the  technic  have  been  right.  It  is  best,  how- 
ever, to  examine  several  drops;  and,  when  the  sediment 
is  abundant,  drops  from  the  upper  and  lower  portions 
should  be  examined  separately. 

In  examining  urinary  sediments  microscopically  no 
fault  is  so  common,  nor  so  fatal  to  good  results,  as  im- 
proper illumination  (see  Fig.  4),  and  none  is  so  easily 
corrected.  The  light  should  be  central  and  very  sub- 
dued for  ordinary  work,  but  oblique  illumination,  ob- 
tained by  swinging  the  mirror  a  little  out  of  the  optical 
axis,  will  be  found  helpful  in  identifying  certain  dehcate 
structures  like  hyaline  casts.  The  16  mm.  objective 
should  be  used  as  a  finder,  while  the  4  mm.  is  reserved 
for  examining  details.  An  experienced  worker  will  rely 
almost  wholly  upon  the  lower  power. 

It  is  well  to  emphasize  that  the  most  common  errors 
which  result  in  failure  to  find  important  structures,  when 
present,  are  lack  of  care  in  transferring  the  sediment  to  the 
slide,  too  strong  illumination,  and  too  great  magnification. 

In  order  to  distinguish  between  similar  structures  it  is 
often  necessary  to  watch  the  effect  upon  them  of  certain 
reagents.  This  is  especially  true  of  the  various  unorgan- 
ized sediments.  They  very  frequently  cannot  be  identi- 
fied from  their  form  alone.  With  the  structures  still  in 
focus,  a  drop  of  the  reagent  may  be  placed  at  one  edge  of 
the  cover-glass  and  drawn  underneath  it  by  the  suction  of 
a  piece  of  blotting-paper  touched  to  the  opposite  edge; 
or  a  small  drop  of  the  reagent  and  of  the  urine  may  be 
placed  close  together  upon  a  slide  and  a  cover  gently 
lowered  over  them.  As  the  two  fluids  mingle,  the  effect 
upon  various  structures  may  be  seen. 


MICROSCOPIC  EXAMINATION 


141 


Urinary  sediments  may  be  studied  under  three  heads: 
A.  Unorganized  sediments.  B.  Organized  sediments.  C. 
Extraneous  structures. 


A.  Unorganized  Sediments 
In  general  these  have  little  diagnostic  or  prognostic 
significance.  Most  of  them  are  substances  normally 
present  in  solution,  which  have  been  precipitated  either 
because  present  in  excessive  amounts,  or,  more  frequently, 
because  of  some  alteration  in  the  urine  (as  in  reaction, 
concentration,  etc.)  which  may  be  purely  physiologic, 
depending  upon  changes  in  diet  or  habits.  Various 
substances  are  always  precipitated  during  decompo- 
sition, which  may  take  place  either  within  or  without 


Fig.  36. — Unusual  urinary  crystals  (drawn  from  various  authors):  i,  Calcium  sul- 
phate (colorless);  2,  cholesterin  (colorless);  3,  hippxiric  acid  (colorless);  4,  hematoidin 
(brown);  5,  fatty  acids  (colorless);  6,  indigo  (blue);  7,  sodium  urate  (yellowish). 

the  body.  Unorganized  sediments  may  be  classified 
according  to  the  reaction  of  the  urine  in  which  they  are 
most  likely  to  be  found : 

In  acid  urine:  Uric  acid,  amorphous  urates,  sodium 
urate,  calcium  oxalate,  leucin  and  ty rosin,  cystin,  and 
fat-globules.     Uric  acid,  the  urates,  and  calcium  oxalate 


142 


THE    URINE 


are  the  common  deposits  of  acid  urines;  the  others  are 
less  frequent,  and  depend  less  upon  the  reaction  of  the 
urine. 

In  alkaline  urine:  Phosphates,  calcium  carbonate,  and 
ammonium  urate. 

Other  crystalline  sediments  (Fig.  36)  which  are  rare 
and  require  no  further  mention  are:  Calcium  sulphate, 
cholesterin,  hippuric  acid,  hematoidin,  fatty  acids,  and 
indigo. 


Fig-  37- — Forms  of  uric  acid:  i,  Rhombic  plates;  2,  whetstone  forms;  3,  3,  quadrate 
forms;  4,  5.  prolonged  into  points;  6,  8,  rosets;  7.  pointed  bundles;  g,  barrel  forms  pre- 
cipitated by  adding  hydrochloric  acid  to  urine  (Ogden). 

I.  In   Acid   Urine.— (i)  Uric-acid    Crystals.— These 

crystals  are  the  red  grains — "  gravel  "  or  "  red  sand  " — 
which  are  often  seen  adhering  to  the  sides  and  bottom 
of  a  vessel  containing  urine.  ]VIicroscopIcally,  they  are 
yellow  or  reddish-brown  crystals,  which  differ  greatly  in 


PLATE  III 


Uric-acid  crystals  with  amorphous  urates  (after  Payer). 


MICROSCOPIC   EXAMINATION  143 

size  and  shape.  The  most  characteristic  forms  (Plate  III 
and  Fig.  37)  are  "whetstones";  roset-like  clusters  of 
prisms  and  whetstones;  and  rhombic  plates,  which 
have  usually  a  paler  color  than  the  other  forms  and  are 
sometimes  colorless.  A  very  rare  form  is  a  colorless 
hexagonal  plate  resembling  cystin.  Recognition  of  the 
crystals  depends  less  upon  their  shape  than  upon  their 
color,  the  reaction  of  the  urine,  and  the  facts  that  they 
are  soluble  in  caustic  soda  solution  and  insoluble  in  hy- 
drochloric or  acetic  acid.  When  ammonia  is  added, 
they  dissolve  and  crystals  of  ammonium  urate  appear. 

A  deposit  of  uric-acid  crystals  has  no  significance  un- 
less it  occurs  before  or  very  soon  after  the  urine  is  voided. 
Every  urine,  if  kept  acid,  will  in  time  deposit  its  uric 
acid.  Factors  which  favor  an  early  deposit  are  high 
acidity,  diminished  urinary  pigments,  and  excessive  ex- 
cretion of  uric  acid.  The  chief  clinical  interest  of  the 
crystals  lies  in  their  tendency  to  form  calculi,  owing  to 
the  readiness  with  which  they  collect  about  any  solid 
object.  Their  presence  in  the  freshly  voided  urine  in 
clusters  of  crystals  suggests  stone  in  the  kidney  or 
bladder,  especially  if  blood  is  also  present.    (See  Fig.  65.) 

(2)  Amorphous  Urates. — These  are  chiefly  urates  of 
sodium  and  potassium  which  are  thrown  out  of  solution 
as  a  yellow  or  red  "  brick-dust  "  deposit.  In  pale 
urines  this  sediment  is  almost  white.  It  disappears  upon 
heating.  A  deposit  of  amorphous  urates  is  very  common 
in  concentrated  and  strongly  acid  urines,  especially  in 
cold  weather,  and  has  no  clinical  significance.  Under 
the  microscope  it  appears  as  fine  yellowish  granules, 
often  so  abundant  as  to  obscure  all  other  structures 
(Plate  III).     In  such  cases  the  urine  should  be  warmed 


144  THE    URINE 

before  examining.  Amorphous  urates  are  readily  sol- 
uble in  caustic  soda  solutions.  When  treated  with  hy- 
drochloric or  acetic  acid,  they  slowly  dissolve  and  rhombic 
crystals  of  uric  acid  appear. 

Rarely,  sodium  urate  occurs  in  crystalline  form — 
slender  prisms,  arranged  in  fan-  or  sheaf-hke  structures 
(Fig.  36). 

(3)  Calcium  Oxalate. — Characteristic  of  calcium  oxa- 
late are  colorless,  glistening,  octahedral  crystals,  giving 
the  appearance  of  small  squares  crossed  by  two  intersect- 


ffi 


^ 


Fig.  38. — Various  forms  of  calcium  oxalate  crystals  (Ogden). 

ing  diagonal  lines — the  so-called  "  envelop  crystals  " 
(Fig.  51).  They  vary  greatly  in  size,  being  sometimes 
so  small  as  to  seem  mere  points  of  light  with  medium- 
power  objectives.  Unusual  forms,  which,  however, 
seldom  occur  except  in  conjunction  with  the  octahedra, 
are  colorless  dumb-bells,  spheres,  and  variations  of  the 
octahedra  (Fig.  38).  The  spheres  might  be  mistaken  for 
globules  of  fat  or  red  blood-corpuscles.  Crystals  of 
calcium  oxalate  are  insoluble  in  acetic  acid  or  caustic 
soda.     They  are  dissolved  by  strong  hydrochloric  acid, 


MICROSCOPIC  EXAMINATION  I45 

and  recrystallize  as  octahedra  upon  addition  of  ammonia. 
They  are  sometimes  encountered  in  alkaline  urine. 

The  crystals  are  commonly  found  in  the  urine  after 
ingestion  of  vegetables  rich  in  oxaHc  acid,  as  tomatoes, 
spinach,  asparagus,  and  rhubarb.  They  have  no  de- 
finite significance  pathologically.  They  often  appear 
in  digestive  disturbances,  in  neurasthenia,  and  when  the 
oxidizing  power  of  the  system  is  diminished.  When 
abundant,  they  are  generally  associated  with  a  little 
mucus;  and,  in  men,  frequently  with  a  few  spermatozoa. 
Like  uric  acid,  their  chief  clinical  interest  lies  in  their 
tendency  to  form  calculi,  and  their  presence  in  fresh 
urine,  together  with  evidences  of  renal  or  cystic  irritation, 
should  be  viewed  with  suspicion,  particularly  if  they  are 
clumped  in  small  masses. 

(4)  Leucin  and  Tyrosin. — Crystals  are  deposited  only 
when  the  substances  are  present  in  considerable  amount. 
When  present  in  smaller  amount,  they  will  usually  be 
deposited  if  a  little  of  the  urine  be  slowly  evaporated  upon 
a  slide.  Addition  of  alcohol  favors  the  deposit.  They 
generally  appear  together,  and  are  of  comparatively 
rare  occurrence,  usually  indicating  severe  fatty  destruc- 
tion of  the  liver,  such  as  occurs  in  acute  yellow  atrophy 
and  phosphorus-poisoning. 

The  crystals  cannot  be  identified  from  their  morphol- 
ogy alone,  since  other  substances,  notably  calcium  phos- 
phate (Fig.  42)  and  ammonium  urate,  may  take  similar 
or  identical  forms. 

Leucin  crystals  (Fig.  39)  as  they  appear  in  the  urine 

do  not  represent  the  pure  substance.     They  are  slightly 

yellow,  oily-looking  spheres,  many  of  them  with  radial 

and  concentric  striations.      Some   may  be   merged  to- 

10 


146  THE   URINE 

gether  in  clusters.  They  are  not  soluble  in  hydrochloric 
acid  nor  in  ether. 

Tyrosin  crystallizes  in  very  fine  colorless  needles, 
usually  arranged  in  sheaves,  with  a  marked  constriction 
at  the  middle  (Fig.  39).  It  is  soluble  in  ammonia  and 
hydrochloric  acid,  but  not  in  acetic  acid. 

(5)  Cystin  crystals  are  colorless,  highly  refractive, 
rather  thick,  hexagonal  plates  with  well-defined  edges. 
They  lie  either  singly  or  superimposed  to  form  more  or 
less  irregular  clusters  (Fig.  40).     Uric  acid  sometimes 


Fig.  3Q. — Leucin  spheres  and  tyrosin  needles  (Stengel). 

takes  this  form  and  must  be  excluded.  Cystin  is  soluble 
in  hydrochloric  acid,  insoluble  in  acetic;  it  is  readily 
soluble  in  ammonia  and  recrystallizes  upon  addition  of 
acetic  acid. 

Cystin  is  one  of  the  amino-acids  formed  in  decompo- 
sition of  the  protein  molecule,  and  is  present  in  traces  in 
normal  urine.  Crystals  are  deposited  only  when  the  sub- 
stance is  present  in  excessive  amount.  Their  presence 
is  known  as  cystiniiria.  It  is  a  rare  condition  due  to  an 
obscure  abnormality  of  protein  metabolism  and  usually 


MICROSCOPIC   EXAMINATION 


147 


continues  throughout  life.  There  are  rarely  any  symp- 
toms save  those  referable  to  renal  or  cystic  calculus,  to 
which  the  condition  strongly  predisposes. 

(6)  Fat-globules. — Fat  appears  in  the  urine  as  highly 
refractive  globules  of  various  sizes,  frequently  very  small. 
These  globules  are  easily  recognized  from  the  fact  that 
they  are  stained  black  by  osmic  acid  and  orange  or  red 
by  Sudan  III.     The  stain  may  be  applied  upon  the  slide, 


Fig.  40. — Cystin  crystals  from  urine  of  patient  with  cystin  calculus  ( X  200)  (photograph  bj 

the  author). 


as  already  described  (p.  140).  Osmic  acid  should  be 
used  in  i  per  cent,  aqueous  solution ;  Sudan  III  in  satu- 
rated solution  in  70  per  cent,  alcohol,  to  which  one-half 
volume  of  10  per  cent,  formalin  may  advantageously 
be  added. 

Fat  in  the  urine  is  usually  a  contamination  from  un- 
clean vessels,  oiled  catheters,  etc.  A  very  small  amount 
may  be  present  after  ingestion  of  large  quantities  of  cod- 


148  THE    URINE 

liver  oil  or  other  fats.  In  fatty  degeneration  of  the 
kidney,  as  in  phosphorus-poisoning  and  chronic  paren- 
chymatous nephritis,  fat-globules  are  commonly  seen, 
both  free  in  the  urine  and  embedded  in  cells  and  tube- 
casts. 

In  chyluria,  or  admixture  of  chyle  with  the  urine  as  a 
result  of  rupture  of  a  lymph-vessel,  minute  droplets  of 
fat  are  so  numerous  as  to  give  the  urine  a  milky  appear- 
ance. The  droplets  are  generally  smaller  than  those  of 
milk.  The  fluid  is  often  blood-tinged.  Chyluria  occurs 
most  frequently  as  a  symptom  of  infection  by  filaria 
(p.  357),  the  embryos  of  which  can  usually  be  found  in 
the  milky  urine. 

2.  In  Alkaline  Urine.— (i)  Phosphates.— While  most 
common  in  alkaline  urine,  phosphates  are  sometimes 
deposited  in  amphoteric  or  feebly  acid  urines.  The  usual 
forms  are:  {a)  Ammoniomagnesium  phosphate  crystals; 
{h)  acid  calcium  phosphate  crystals;  and  (c)  amorphous 
phosphates.     All  are  readily  soluble  in  acetic  acid. 

{a)  Ammonioynagnesium  Phosphate  Crystals. — They 
are  the  common  "  triple  phosphate  "  crystals,  which  are 
generally  easily  recognized  (Figs.  41  and  66.  and  Plate 
IV).  They  are  colorless,  except  when  bile-stained. 
Their  usual  form  is  some  modification  of  the  prism,  with 
oblique  ends.  Alost  tx-pical  are  the  well-known  '*  coffin- 
Hd  "  and  ''  hip-roof  "  forms.  The  long  axis  of  the  hip- 
roof crystal  is  often  so  shortened  that  it  resembles  the 
envelop  crystal  of  calcium  oxalate.  It  does  not,  how- 
ever, have  the  same  luster;  this,  and  its  solubility  in  acetic 
acid,  will  always  prevent  confusion. 

When  rapidly  deposited,  as  by  artificial  precipitation, 
triple  phosphate  often  takes  feathery,  star-,  or  leaf-like 


MICROSCOPIC  EXAMINATION 


149 


forms.  These  gradually  develop  into  the  more  common 
prisms.  X-forms  may  be  produced  by  partial  solution  of 
prisms. 

(b)  Acid  Calcium  Phosphate  Crystals.- — In  feebly  acid, 
amphoteric,  or  feebly  alkaline  urines  acid  calcium  phos- 
phate, wrongly  called  "  neutral  calcium  phosphate," 
is  not  infrequently  deposited  in  the  form  of  colorless 
prisms  arranged  in  stars  and  rosets  (Fig.  42,  i).  The 
individual  prisms  are  usually  slender,  with  one  beveled, 


Fig.  41. — Various  forms  of  triple  phosphate  crystals  (Ogden). 

wedge-like  end,  but  are  sometimes  needle-like.  They 
may  sometimes  take  forms  resembling  tyrosin  (Fig.  42, 
2),  calcium  sulphate,  or  hippuric  acid,  but  are  readily 
distinguished  by  their  solubility  in  acetic  acid. 

Calcium  phosphate  often  forms  large,  thin,  irregular, 
usually  granular,  colorless  plates,  which  are  easily  recog- 
•nized  (Fig.  42,  3). 

(c)  Amorphous  Phosphates. — The  earthy  phosphates 
are  thrown  out  of  solution  in  most  alkaline  and  many 
amphoteric   urines   as   a   white,    amorphous   sediment, 


150 


THE    URINE 


which  may  be  mistaken  for  pus  macroscopically.  Under 
the  microscope  the  sediment  is  seen  to  consist  of  numer- 
ous colorless  granules,   distinguished   from  amorphous 


V 


Fig.  42. — Crystals  of  calcium  phosphate:  i,  Common  form  Ccopied  from  Rieder's 
Atlas);  2,  needles  resembling  tyrosin  (drawn  from  nature);  3,  large,  irregular  plates  ({rem 
nature). 

urates  by  their  color,  their  solubility  in  acetic  acid,  and 
the  reaction  of  the  urine. 

The  various  phosphatic  deposits  frequently  occur 
together.     They  are  sometimes  due  to  excessive  excre- 


9=  '     ff'^%^ 


•^  .^^^ 


^'^^ 


*d? 


Fig.  43. — Indistinct  crystalline  sediment   (dumb-bell  crystals)  of  calcium  carbonate. 
Similar  crystals  are  formed  by  calcium  oxalate  and  calcium  sulphate  (after  Funke). 

tion  of  phosphoric  acid,  but  usually  merely  indicate  that 
the  urine  has  become,  or  is  becoming,  alkaline.  (See 
Phosphates,  p.  86.) 

(2)  Calcium   carbonate   may  sometimes  be  mingled 
with   the   phosphatic   deposits,   usually   as  amorphous 


PLATE  IV 


Sediment   of   alkaline   fermentation   (after   Hofmann   and    Ultzmann). 


MICROSCOPIC   EXAMINATION  151 

granules,  or,  more  rarely,  as  -cfelorl^sg^Sjph^res  ^nd  dumb- 
bells (Fig.  43),  which  are  soXu\:)^(^kh  9x^)Ac  acici  wi'tli  ^^s/*.  Tii 
formation.  '   '      '   ^  '  ^  '   '  ff« 

(3)  Ammonium  Urate  Crystals. — This  is  the  only 
urate  deposited  in  alkaline  urine.  It  forms  opaque 
yellow  crystals,  usually  in  the  form  of  spheres  (Plate  IV. 
and  Fig.  66) ,  which  are  often  covered  with  fine  or  coarse 


Fig.  44.— Crystals  of  ammonium  urate  (one-half  of  the  forms  copied  from  Rieder's  Atlas, 
the  others  from  nature). 

spicules — "  thorn-apple  crystals."  Sometimes  dumb- 
bells, compact  sheaves  of  fine  needles,  and  irregular 
rhizome  forms  are  seen  (Fig.  44).  Upon  addition  of 
acetic  acid  they  dissolve,  and  rhombic  plates  of  uric  acid 
appear. 

These  crystals  occur  only  when  free  ammonia  is 
present.  They  are  generally  found  along  with  the  phos- 
phates in  decomposing  urine  and  have  no  clinical 
significance. 

B.     Organized  Sediments 
The  principal  organized  structures  in  urinary  sedi- 
ments are:   Tube-casts;  epithelial  cells;  pus-corpuscles; 


152  THE    URINE 

red  bIood-corpusde>^ ;  sj^ennatozoa ;  bacteria,  and  animal 
parasites.  They  are  njuck  more  important  than  the 
unorganized  sediments  just  considered. 

1 .  Tube=casts. — These  interesting  structures  are  albu- 
minous casts  of  the  uriniferous  tubules.  Their  pres- 
ence in  the  urine  probably  always  indicates  some 
pathologic  change  in  the  kidney,  although  this  change 
may  be  very  slight  or  transitory.  Large  numbers  may 
be  present  in  temporary  irritations  and  congestions. 
They  do  not  in  themselves,  therefore,  imply  organic  dis- 
ease of  the  kidney.  They  rarely  occur  in  urine  which 
does  not  contain,  or  has  not  recently  contained,  al- 
bumin. 

While  it  is  not  possible  to  draw  a  sharp  dividing-line 
between  the  different  varieties,  casts  may  be  classified 
as  follows: 

(i)  Hyaline  casts. 

(a)  Narrow. 

(b)  Broad. 

(2)  Waxy  casts. 

(3)  Fibrinous  casts. 

(4)  Granular  casts. 

(a)  Finely  granular. 
(h)   Coarsely  granular. 

(5)  Fatty  casts. 

(6)  Casts  containing  organized  structures. 

((/)  Epithelial  casts. 

(b)  Blood-casts. 

(c)  Pus-casts. 

(d)  Bacterial  casts. 

As  will  be  seen  later,  practically  all  varieties  are 
modifications  of  the  hyaline. 


MICROSCOPIC  EXAMINATION  1 53 

The  significance  of  the  different  varieties  is  more 
readily  understood  if  one  considers  their  mode  of  forma- 
tion. Albuminous  material,  the  source,  and  nature  of 
which  are  not  definitely  known,  but  which  are  doubtless 
not  the  same  in  all  cases,  probably  enters  the  lumen  of  a 
uriniferous  tubule  in  a  fluid  or  plastic  state.  The 
material  has  been  variously  thought  to  be  an  exudate 
from  the  blood,  a  pathologic  secretion  of  the  renal  cells, 
and  a  product  of  epithelial  degeneration.  In  the  tubule 
it  hardens  into  a  cast  which,  when  washed  out  by  the 
urine,  retains  the  shape  of  the  tubule,  and  contains  within 
its  substance  whatever  structures  and  debris  were  lying 
free  within  the  tubule  or  were  loosley  attached  to  its  wall. 
If  the  tubule  be  small  and  have  its  usual  lining  of  epithe- 
lium, the  cast  will  be  narrow;  if  it  be  large  or  entirely 
denuded  of  epithelium,  the  cast  will  be  broad.  A  cast, 
therefore,  indicates  the  condition  oj  the  tubule  in  which 
it  is  formed,  but  does  not  necessarily  indicate  the  condition 
of  the  kidney  as  a  whole. 

The  search  for  casts  must  be  carefully  made.  The 
urine  must  be  fresh,  since  hyaline  casts  soon  dissolve 
when  it  becomes  alkaline.  It  should  be  thoroughly 
centrifugalized.  When  the  sediment  is  abundant,  casts, 
being  light  structures,  will  be  found  near  the  top.  In 
cystitis,  where  casts  may  be  entirely  hidden  by  the  pus, 
the  bladder  should  be  irrigated  to  remove  as  much  of 
the  pus  as  possible  and  the  next  urine  examined.  In 
order  to  prevent  solution  of  the  casts  the  urine,  if  al- 
kaline, must  be  rendered  acid  by  previous  administra- 
tion of  boric  acid  or  other  drugs.  Heavy  sediments  of 
urates,  blood,  or  vaginal  cells  may  likewise  obscure 
casts  and  other  important  structures.     The  last  can  be 


154 


THE    URINE 


avoided  by  catheterization.  Urates  can  be  dissolved 
by  gently  warming  before  centrifugalizing,  care  being 
taken  not  to  heat  enough  to  coagulate  the  albumin. 
The  albumin  shield  of  .the  centrifuge  tube  may  also 
be  heated.  Blood  can  be  destroyed  by  centrifugalizing, 
pouring  off  the  supernatant  urine,  tilling  the  tube  with 
water,  adding  a  few  drops  of  dilute  acetic  acid,  mixing 
well,  and  again  centrifugalizing;  this  process  being 
repeated  until  the  blood  is  completely  decolorized. 
Too  much  acetic  acid  will  dissolve  hyaline  casts. 

Their  cylindric  shape  can  be  best  seen  by  slightly 
moving  the  cover-glass  w^hile  observing  them,  thus 
causing  them  to  roll.  This  little  manipulation  should 
be  practised  until  it  can  be  done  satisfactorily.  It  will 
prove  useful  in  many  examinations. 

Various  methods  of  staining  casts  so  as  to  render  them 
more  conspicuous  have  been  proposed.  They  offer  no 
special  advantage  to  one  who  understands  how  to  use 
the  substage  mechanism  of  his  microscope.  The  "  nega- 
tive-staining ■'  method  is  as  good  as  any.  It  consists 
simply  in  adding  a  little  India-ink  to  the  drop  of  urine  on 
the  slide.  Casts,  cells,  etc.,  will  stand  out  as  colorless 
structures  on  a  dark  background. 

(i)  Hyaline  Casts. — Typically,  these  are  colorless, 
homogeneous,  semitransparent,  cyhndric  structures,  with 
parallel  sides  and  usually  rounded  ends.  Not  infre- 
quently they  are  more  opaque  or  show  a  few  granules  or 
an  occasional  oil-globule  or  cell,  either  adhering  to  them 
or  contained  within  their  substance.  Generally  they 
are  straight  or  curved;  less  commonly,  convoluted. 
Their  length  and  breadth  vary  greatly :  they  are  some- 
times so  long  as  to  extend  across  several  fields  of  a 


MICROSCOPIC  EXAMINATION 


155 


medium-power  objective,  but  are  usually  much  shorter; 
in  breadth,  they  vary  from  one  to  seven  or  eight  times 


Fig.  45. — Hyaline  casts  showing  fat-droplets  and  leukocytes  (obj.  one-sixth)  (Boston). 


the  diameter  of  a  red  blood-corpuscle.     (See  Figs.  4,  45, 
46,  and  50.) 


Fig.  46. — Various  kinds  of  casts:  a.  Hyaline  and  finely  granular  cast;  b,  finely  granular 
cast;  c,  coarsely  granular  cast;  d,  brown  granular  cast;  e,  granular  cast  with  normal  and 
abnormal  blood  adherent;  /,  granular  cast  with  renal  cells  adherent;  ;;,  granular  cast  with 
fat  and  a  fatty  renal  cell  adherent  COgden). 

Hyaline  casts  are  the  least  significant  of  all  the  casts, 
and  occur  in  many  slight  and   transitory  conditions. 


156  THE   URINE 

Small  numbers  are  common  following  ether  anesthesia, 
in  fevers,  after  excessive  exercise,  and  in  congestions  and 
irritations  of  the  kidney.  They  are  always  present,  and 
are  usually  stained  yellow  when  the  urine  contains  much 
bile.  While  they  are  found  in  all  organic  diseases  of  the 
kidney,  they  are  most  important  in  chronic  interstitial 
nephritis.  Here  they  are  seldom  abundant,  but  their 
constant  presence  is  the  most  reliable  urinary  sign  of  the 
disease.  Small  areas  of  chronic  interstitial  change  are 
probably  responsible  for  the  few  hyaline  casts  so  fre- 
quently found  in  the  urine  of  elderly  persons. 


Fig.  47. — Waxy  casts  (upper  part  of  figure).     Fatty  and  fat-bearing  casts  (lower  part  of 
figure)  (from  Greene's  "Medical  Diagnosis"). 

Very  broad  hyaline  casts  commonly  indicate  complete 
desquamation  of  the  tubular  epithelium,  such  as  occurs 
in  the  late  stages  of  nephritis. 

(2)  Waxy  Casts. — Like  hyaline  casts,  these  are  homo- 
geneous when  typical,  but  frequently  contain  a  few 
granules  or  an  occasional  cell.  They  are  much  more 
opaque  than  the  hyaline  variety,  and  are  usually  shorter 
and  broader,  with  irregular,  broken  ends,  and  some- 
times appear  to  be  segmented.  They  are  grayish  or 
colorless,  and  have  a  dull,  waxy  look,  as  if  cut  from  par- 


MICROSCOPIC  EXAMINATION 


157 


aflSn  (Figs.  47  and  64).  They  are  sometimes  composed 
of  material  which  gives  the  amyloid  reactions.  Waxy 
casts  are  found  in  most  advanced  cases  of  nephritis, 
where  they  are  an  unfavorable  sign.  They  are  perhaps 
most  frequently  found  in  amyloid  disease  of  the  kidney, 
but  are  not  distinctive  of  the  disease,  as  is  sometimes 
stated. 

(3)  Fibrinous  Casts. — Casts  which  resemble  waxy 
casts,  but  have  a  distinctly  yellow  color,  as  if  cut  from 
beeswax,  are  often  seen  in  acute  nephritis.     They  are 


Fig.  48. — Granular  and  fatty  casts  and  two  compound  granular  cells  (Stengel). 

called  fibrinous  casts,  but  the  name  is  inappropriate,  as 
they  are  not  composed  of  fibrin.  They  are  often  classed 
with  waxy  casts,  but  should  be  distinguished,  as  their 
significance  is  much  less  serious. 

(4)  Granular  Casts. — These  are  merely  hyaline  casts 
in  which  numerous  granules  are  embedded  (Figs.  46,  48, 
and  50). 

Finely  granular  casts  contain  many  fine  granules,  are 
usually  shorter,  broader,  and  more  opaque  than  the 
hyaline  variety,  and  are  more  conspicuous.  Their  color 
is  grayish  or  pale  yellow. 


158  THE   URINE 

Coarsely  granular  casts  contain  larger  granules  and  are 
darker  in  color  than  the  finely  granular,  being  often  dark 
brown  owing  to  presence  of  altered  blood-pigment.  They 
are  usually  shorter  and  more  irregular  in  outline,  and 
more  frequently  have  irregularly  broken  ends. 

(5)  Fatty  Casts. — Small  droplets  of  fat  may  at  times 
be  seen  in  any  variety  of  cast.  Those  in  which  the  drop- 
lets are  numerous  are  called  fatty  casts  (Figs.  47  and  48) . 
The  fat-globules  are  not  difficult  to  recognize.  Staining 
with  osmic  acid  or  Sudan  (p.  147)  will  remove  any  doubt 
as  to  their  nature. 

The  granules  and  fat-droplets  seen  in  casts  are  prod- 
ucts of  epithelial  degeneration.  Granular  and  fatty 
casts,  therefore,  always  indicate  partial  or  complete  dis- 
integration of  the  renal  epithelium.  The  finely  granular 
variety  is  the  least  significant,  and  is  found  when 
the  epithelium  is  only  moderately  affected.  Coarsely 
granular,  and  especially  fatty  casts,  if  present  in  con- 
siderable numbers,  indicate  a  serious  parenchymatous 
nephritis. 

(6)  Casts  Containing  Organized  Structures. — Cells 
and  other  structures  are  frequently  seen  adherent  to  a 
cast  or  embedded  within  it.  (See  Figs.  45  and  46). 
When  numerous,  they  give  name  to  the  cast. 

(a)  Epithelial  casts  contain  epithelial  cells  from  the 
renal  tubules.  They  always  imply  desquamation  of 
epithelium,  which  rarely  occurs  except  in  parenchy- 
matous inflammations  (Figs.  63  and  64).  When  the 
cells  are  well  preserved  they  point  to  acute  nephritis. 

ib)  Blood-casts  contain  red  blood-corpuscles,  usually 
much  degenerated  (Figs.  49  and  63).  They  always 
indicate  hemorrhage  into  the  tubules,  which  is  most 


MICROSCOPIC  EXAMINATION 


159 


common  in  acute  nephritis  or  an  acute  exacerbation  of 
a  chronic  nephritis. 

(c)  Pus-casts  (see  Fig.  65),  composed  almost  wholly  of 
pus-corpuscles,  are  uncommon,  and  point  to  a  chronic 
suppurative  process  in  the  kidney. 

(d)  True  bacterial  casts  are  rare.  They  indicate  a 
septic  condition  in  the  kidney.  Bacteria  may  permeate 
a  cast  after  the  urine  is  voided. 


Fig.  49. — Red  blood-corpuscles  and  blood-casts  (courtesy  of  Dr.  A.  Scott)  (obj.  one- 
sixth)  (Boston) 

Structures  Likely  to  be  Mistaken  for  Casts. — (i) 
Mucous  Threads. — Mucus  frequently  appears  in  the 
form  of  long  strands  which  slightly  resemble  hyaline 
casts  (Fig.  50).  They  are,  however,  more  ribbon-like, 
have  less  well-defined  edges,  and  usually  show  faint 
longitudinal  striations.  Their  ends  taper  to  a  point  or 
are  split  or  curled  upon  themselves,  and  are  never  evenly 
rounded,  as  is  commonly  the  case  with  hyaline  casts. 

Such  threads  form  a  part  of  the  nubecula  of  normal 
urine,  and  are  especially  abundant  when  calcium  oxalate 


i6o 


THE    URINE 


crystals  are  present.  When  there  is  an  excess  of  mucus, 
as  in  irritations  of  the  urinary  tract,  every  field  may  be 
filled  with  an  interlacing  meshwork. 

Mucous  threads  are  microscopic  and  should  not  be 
confused  with  urethral  shreds,  which  are  macroscopic, 
and  consist  of  a  matrix  of  mucus  in  which  many  epi- 
thelial and  pus-cells  are  embedded. 

(2)  Cylindroids. — This  name  is  sometimes  given  to  the 
mucous  threads  just  described,  but  is  more  properly 


Fig.  so. — Hyaline  and  granular  casts,  mucous  threads,  and  cylindroids.    There  are  also 
a  few  epithelial  cells  from  the  bladder  (Wood). 

applied  to  certain  peculiar  structures  more  nearly  allied 
to  casts.  They  resemble  hyaline  casts  in  structure,  but 
differ  in  being  broader  at  one  end  and  tapering  to  a 
slender  tail,  which  is  often  twisted  or  curled  upon  itself 
(Fig.  50).  They  frequently  occur  in  the  urine  along 
with  hyaline  casts,  especially  in  irritations  of  the  kidney, 
and  have  no  definite  pathologic  significance. 

(3)  Masses  of  amorphous  urates,  or  phosphates,  or 


MICROSCOPIC  EXAMINATION  l6l 

very  small  crystals  (Fig.  51),  which  accidentally  take  a 
cylindric  form,  or  shreds  of  mucus  covered  with  granules, 
closely  resemble  granular  casts.  Application  of  gentle 
heat  or  appropriate  chemicals  will  serve  to  differentiate 
them.  When  urine  contains  both  mucus  and  granules, 
large  numbers  of  these  "  pseudocasts,"  all  lying  in  the 
same  direction,  can  be  produced  by  slightly  moving  the 
cover-glass  from  side  to  side.  It  is  possible — as  in  urate 
infarcts  of  infants — for  urates  to  be  molded  into  cylin- 
dric bodies  within  the  renal  tubules. 


Fig.  SI. — Calcium  oxalate  crystals,  showing  a  pseudocast  of  small  crystals  (Jakob). 

(4)  Hairs  and  fibers  of  wool,  cotton,  etc.  These 
could  be  mistaken  for  casts  only  by  beginners.  One 
can  easily  become  familiar  with  their  appearance  by 
suspending  them  in  water  and  examining  with  the  micro- 
scope (Fig.  61). 

(5)  Hyphae  of  molds  are  not  infrequently  mistaken 
for  hyaline  casts.  Their  higher  degree  of  refraction, 
their  jointed  or  branching  structure,  and  the  accom- 
panying spores  will  differentiate  them  (Fig.  62). 

11 


1 62 


THE   URINE 


2.  Epithelial  Cells.— A  few  cells  from  various  parts 
of  the  urinary  tract  occur  in  every  urine.  A  marked 
increase  indicates  some  pathologic  condition  at  the  site  of 
their  origin.  It  is  sometimes,  but  by  no  means  always, 
possible  to  locate  their  source  from  their  form.  Most 
cells  are  much  altered  from  their  original  shape.  Any 
epithelial  cell  may  be  so  granular  from  degenerative 
changes  that  the  nucleus  is  obscured.  They  are  usually 
divided  into  three  groups: 

(i)  Small,  round  or  polyhedral 
^mW^£f  cells  are  about  the  size  of   pus- 

^k^m  corpuscles,  or  a  little  larger,  with  a 

v^tfft  «...       single  round  nucleus.      Such  cells 

may  come  from  the  deeper  layers 
of  any  part  of  the  urinary  tract. 
They  are  uncommon  in  normal 
urine.  When  they  are  dark  in 
color,  very  granular,  and  contain 
a  comparatively  large  nucleus,  they 
probably  come  from  the  renal  tub- 
ules, but  their  origin  in  the  kid- 
ney is  not  proved  unless  they  are 
found  embedded  in  casts.  Renal 
cells  are  abundant  in  parenchyma- 
tous nephritis,  especially  the  acute  form.  They  are 
nearly  always  fatty — most  markedly  so  in  chronic  paren- 
chymatous nephritis,  where  their  substance  is  sometimes 
wholly  replaced  by  fat-droplets  ("  compound  granule 
cells  ")  (see  Figs.  48,  52,  and  63). 

(2)  Irregular  cells  are  considerably  larger  than  the 
preceding.  They  are  round,  pear  shaped,  or  spindle 
shaped,  or  may  have  tail-like  processes,  and  are  hence 


Fig.  S2- — Renal  epithelium 
from  nephritic  urine:  a,  Poly- 
hedral epithelium  in  nephritis 
of  scarlet  fever;  b  and  c,  differ- 
ent grades  of  fatty  degenera- 
tion in  renal  epithelium  in 
chronic  nephritis  ( X  400)  (after 
Bizzozero). 


MICROSCOPIC  EXAMINATION 


163 


named  large  round,  pyriform,  spindle,  or  caudate  cells 
respectively.  Each  contains  a  round  or  oval  distinct 
nucleus.  Their  usual  source  is  the  deeper  layers  of  the 
urinary  tract,  especially  of  the  bladder.  Caudate  forms 
come  most  commonly  from  the  pelvis  of  the  kidney  (see 
Figs.  53,  h,  54,  65,  and  66). 

(3)  Squamous  or  pavement  cells  are  large  flat  cells, 
each  with  a  small,  distinct,  round  or  oval  nucleus  (Fig. 
53,  a).    They  are  derived  from  the  superficial  layers  of 


Fig-  53- — Epithelial  cells  from  urethra  and  bladder:  a.  Squamous  cells  from  superficia' 
layers;  b,  irregular  cells  from  deeper  layers  (Jakob). 


the  ureters,  bladder,  urethra,  or  vagina,  and  when 
desquamation  is  active,  appear  in  stratified  masses. 
Squamous  cells  from  the  bladder  are  generally  rounded, 
while  those  from  the  vagina  are  larger,  thinner,  and 
more  angular.  Great  numbers  of  these  vaginal  cells, 
"together  with  pus-corpuscles,  may  be  present  when 
leukorrhea  exists. 

3.  Pus=corpuscles.— A  very  few  leukocytes  are  pres- 
ent in  normal  urine.     They  are  more  abundant  when 


i64 


THE    URINE 


mucus  is  present.  An  excess  of  leukocytes,  mainly  of 
the  polymorphonuclear  variety,  with  albumin,  consti- 
tutes pyuria — pus  in  the  urine. 


Fig.  54. — Caudate  epithelial  cells  from  pelvis  of  kidney  (Jakob). 

When  at  all  abundant,  pus  forms  a  w^iite  sediment 
resembling  amorphous  phosphates  macroscopically.    Un- 


®  ®  ® 
® 


®   ®  ® 


Fig-  55- — Pus<orpuscles:  a,  .\s  ordinarily  seen;  h,  ameboid  corpuscles;  c,  showing  the 
action  of  acetic  acid  (Ogden). 


der  the  microscope  the  corpuscles  appear  as  very  granu- 
lar cells,  about  twice  the  diameter  of  a  red  blood-cor- 
puscle (Figs.  55  and  66).     In  freshly  voided  urine  many 


MICROSCOPIC  EXAMINATION  1 65 

exhibit  ameboid  motion,  assuming  irregular  outlines. 
Each  contains  one  irregular  nucleus  or  several  small, 
rounded  nuclei.  The  nuclei  are  obscured  or  entirely 
hidden  by  the  granules,  but  may  be  brought  clearly 
into  view  by  running  a  httle  acetic  acid  under  the  cover- 
glass.  This  enables  one  to  easily  distinguish  pus-cor- 
puscles from  small  round  epithelial  cells,  which  resemble 
them  in  size,  but  have  a  single,  rather  large,  round 
nucleus.  In  decomposing  urine  pus  is  converted  into  a 
gelatinous  mass  which  gives  the  urine  a  ropy  consistence. 

Pyuria  indicates  suppuration  in  some  part  of  the 
urinary  tract — urethritis,  cystitis,  pyelitis,  etc. — or  may 
be  due  to  contamination  from  the  vagina,  in  which  case 
many  vaginal  epithelial  cells  will  also  be  present.  In 
general,  the  source  of  the  pus  can  be  determined  only  by 
the  accompanying  structures  (epithelia,  casts)  or  by  the 
clinical  signs. 

A  fairly  accurate  idea  of  the  quantity  of  pus  from  day 
to  day  may  be  had  by  shaking  the  urine  thoroughly  and 
counting  the  number  of  corpuscles  per  cubic  millimeter 
upon  the  Thoma-Zeiss  blood-counting  slide. 

4.  Red  Blood=corpuscles.— Urine  which  containsblood 
is  always  albuminous.  Very  small  amounts  do  not  alter 
its  macroscopic  appearance.  Larger  amounts  alter  it 
considerably.  Blood  from  the  kidneys  is  generally 
intimately  mixed  with  the  urine  and  gives  it  a  hazy 
reddish  or  brown  color.  When  from  the  lower  urinary 
tract,  it  is  not  so  intimately  mixed  and  settles  more 
quickly  to  the  bottom,  the  color  is  brighter,  and  small 
clots  are  often  present. 

Red  blood-corpuscles  are  not  usually  difficult  to  recog- 
nize with  the  microscope.     When  very  fresh,  they  have  a 


1 66  THE   URINE 

normal  appearance,  being  yellowish  discs  of  uniform  size 
(normal  blood).  When  they  have  been  in  the  urine  any 
considerable  time,  their  hemoglobin  may  be  dissolved  out, 
and  they  then  appear  as  faint  colorless  circles  or  "  shadow 
cells  "  (abnormal  blood),  and  are  more  difficult  to  see 
(Fig.  56;  see  also  Figs.  49  and  63).  They  are  apt  to  be 
swollen  in  dilute  and  crenated  in  concentrated  urines. 
The  microscopic  findings  may  be  corroborated  by  chemic 
tests  for  hemoglobin,  although  the  microscope  may  show 
a  few  red  corpuscles  when  the  chemic  tests  are  negative. 
When  not  due  to  contamination  from  menstrual  dis- 
charge, blood  in  the  urine,  or  hematuria,  is  always  patho- 


O  ^     O  o         o      - 


Fig.  56. — Blood-corpuscles:  a,  Normal;  h,  abnormal  (Ogden). 

logic.  Blood  comes  from  the  kidney  tubules  in  severe 
h^^peremia,  in  acute  nephritis  and  acute  exacerbations  of 
chronic  nephritis,  and  in  renal  tuberculosis  and  malig- 
nant disease.  An  "  idiopathic  hematuria,"  probably  of 
nervous  origin,  has  been  observed.  The  finding  of  blood- 
casts  is  the  only  certain  means  of  diagnosing  the  kidney 
as  its  source.  Blood  comes  from  the  pelvis  of  the  kidney 
in  renal  calculus  (Fig.  65),  and  is  then  usually  intermit- 
tent, small  in  amount,  and  accompanied  by  a  Httle  pus 
and  perhaps  crystals  of  the  substance  forming  the  stone. 
Considerable  hemorrhages  from  the  bladder  may  occur 
in  vesical  calculus,  tuberculosis,  and  new  growths. 
Small   amounts  of   blood   generally   accompany   acute 


MICROSCOPIC   EXAMINATION 


167 


cystitis.  In  Africa  the  presence  of  Schistosomum  hema- 
tobium  in  the  veins  of  the  bladder  is  a  common  cause  of 
hemorrhage  (Egyptian  hematuria) . 

5.  Spermatozoa  are  generally  present  in  the  urine  of 
men  after  nocturnal  emissions,  after  epileptic  convul- 
sions, and  in  spermatorrhea.  They  may  be  found  in  the 
urine  of  both  sexes  following  coitus.  They  are  easily 
recognized  from  their  characteristic  structure  (Fig.  57). 


r  "^ 

y 

0 

© 

^ 

r. 

0 

(, 

J 
i 

?i 

C 

c 
0 

9 

p 

1 
^^ 

Fig.  S7- — Sjjermatozoa  in  urine  (Ogden). 


The  4  mm.  objective  should  be  used,  with  subdued  light 
and  careful  focusing. 

6.  Bacteria. — Normal  urine  is  free  from  bacteria  in 
the  bladder,  but  becomes  contaminated  in  passing 
through  the  urethra.  Various  non-pathogenic  bacteria, 
notably  Micrococcus  urece  (Fig.  58),  are  always  present 
in  decomposing  urine.  In  suppurations  of  the  urinary 
tract  pus-producing  organisms  may  be  found.     In  many 


1 68  THE   URINE 

infectious  diseases  the  specific  bacteria  may  be  eliminated 
in  the  urine  without  producing  any  local  lesion.  Ty- 
phoid bacilli  have  been  known  to  persist  for  months 
and  even  years  after  the  attack. 

Bacteria  produce  a  cloudiness  which  will  not  clear 
upon  filtration.  They  are  easily  seen  with  the  4  mm. 
objective  in  the  routine  microscopic  examination. 
Ordinarily,  no  attempt  is  made  to  identify  any  but  the 
tubercle  bacillus  and  the  gonococcus. 


Fig.  58. — Micrococcus  urese  (after  von  Jaksch). 

Tubercle  bacilli  are  nearly  always  present  in  the  urine 
when  tuberculosis  exists  in  any  part  of  the  urinary  tract, 
but  are  often  difficult  to  find,  especially  when  the  urine 
contains  little  or  no  pus. 


Detection  of  Tubercle  Bacilli  in  Urine. — The  urine  should 
be  obtained  by  catheter  after  careful  cleansing  of  the  parts. 

(i)  Centrifugalize  thoroughly,  after  dissolving  any  sediment 
of  urates  or  phosphates  by  gentle  heat  or  acetic  add.  Pour 
off  the  supernatant  fluid,  add  water,  and  centrifugalize  again. 
Addition  of  one  or  two  volumes  of  alcohol  will  favor  cen- 
trifugalization  by  lowering  the  specific  gravity. 

(2)  Make  thin  smears  of  the  sediment,  adding  a  little  egg- 
albumen  if  necessary  to  make  the  smear  adhere  to  the  glass; 
dry.  and  fix  in  the  usual  way. 

(3)  Stain  with  carbol-fuchsin,  steaming  for  at  least  three 
minutes,  or  at  room  temperature  for  six  to  twelve  Hours. 


■^ 


PLATE  V 


% 
% 


/)  It 


Tubercle  bacilli  in  urinary  sediment;  X  800  (Ogden). 


MICROSCOPIC  EXAMINATION  1 69 

(4)  Wash  in  water,  and  then  in  20  per  cent,  nitric  acid 
until  only  a  faint  pink  color  remains. 

(5)  Wash  in  water. 

(6)  Soak  in  alcohol  fifteen  minutes  or  longer.  This  decolor- 
izes the  smegma  bacillus  (p.  53),  which  is  often  present  in 
the  urinCj^  and  might  easily  be  mistaken  for  the  tubercle  bacil- 
lus. It  is  unlikely,  however,  to  be  present  in  catheterized 
specimens.  It  is  always  safest  to  soak  the  smear  in  alcohol 
for  several  hours  or  over  night,  since  some  strains  of  the  smeg- 
ma bacillus  are  very  resistant. 

(7)  Wash  in  water. 

(8)  Apply  Loffler's  methylene-blue  solution  one-half  minute. 

(9)  Rinse  in  water,  dry  between  filter-papers,  and  examine 
with  the  one-twelfth  objective. 

When  the  bacilli  are  scarce,  the  following  method  may  be 
tried.  It  is  applicable  also  to  other  fluids.  If  the  fluid  is  not 
albuminous,  add  a  little  egg-albumen.  Coagulate  the  albu- 
men by  gentle  heat  and  centrifugalize.  The  bacilli  will  be 
carried  down  with  the  albumen.  The  sediment  is  then  treated 
by  the  antiformin  method  (p.  52). 

A  careful  search  of  many  smears  may  be  necessary  to  find 
the  bacilli.  They  usually  lie  in  clusters  (see  Plate  V).  Fail- 
ure to  find  them  in  suspicious  cases  should  be  followed  by 
inoculation  of  guinea-pigs;  this  is  the  court  of  last  appeal, 
and  must  also  be  sometimes  resorted  to  in  order  to  exclude 
the  smegma  bacillus. 

In  gonorrhea  gonococci  are  sometimes  found  in 
the  sediment,  but  more  commonly  in  the  "  gonorrheal 
threads,"  or  "  floaters."  In  themselves,  these  threads 
are  by  no  means  diagnostic  of  gonorrhea.  Detection  of 
the  gonococcus  is  described  later  (p.  369). 

7.  Animal  parasites  are  rare  in  the  urine.  Booklets 
and  scolices  of    Tcenia  echinococcus  (Fig.  59)  and  em- 


170 


THE    URINE 


bryos  pf  filariae  have  been  met.      In  Africa  the  ova,  and 
even  adults,  of  Schistosomum  hcemalobium  are  common, 


Fig.  5g. — I,  Scolcx  of  ta-nia  cchinococcus.  showin;;  crown  of  booklets;  2,  scolex  and 
detached  booklets  (obj.  one-sixth)  (Boston). 


accompanying    "  Egyptian    hematuria."      Trichomonas 
vaginalis  is  a  not  uncommon  contamination.     This  and 


Fig.  60. — Embryo  of  "vinegar  eel"  in  urine,  from  contamination;  length.  340  Mi 
width,  IS  ix.  .\n  epithelial  cell  from  bladder  and  three  leukocytes  are  also  shown  (studied 
with  Dr.  J.  .\   Wilder). 

other  protozoa  may  be  mistaken  for  spermatozoa  by  the 
inexperienced. 


MICROSCOPIC  EXAMINATION  171 

A  worm  which  is  especially  interesting  is  Anguillula 
aceti,  the  "vinegar  eel."  This  is  generally  present  in 
the  sediment  of  table  vinegar,  and  may  reach  the  urine 
through  use  of  vinegar  in  vaginal  douches,  or  through 
contamination  of  the  bottle  in  which  the  urine  is  con- 
tained. It  has  been  mistaken  for  Sirongyloides  intes- 
tinalis  and  for  the  filaria  embryo.  It  closley  resembles 
the  former  in  both  adult  and  embryo  stages.  The  young 
embryos  have  about  the  same  length  as  filaria  embryos, 
but  are  nearly  twice  as  broad  and  the  intestinal  canal  is 
easily  seen  (compare  Figs.  60  and  134).  For  fuller  de- 
scriptions of  these  parasites  the  reader  is  referred  to 
Chapter  VI. 

C.    Extraneous  Structures 

The  laboratory  worker  must  familiarize  himself  with 
the  microscopic  appearance  of  the  more  common  of  the 
numerous  structures  which  may  be  present  from  acci- 
dental contamination  (Fig.  61). 

Yeast-cells  are  smooth,  colorless,  highly  refractive, 
spheric  or  ovoid  cells.  They  sometimes  reach  the  size  of 
a  leukocyte,  but  are  generally  smaller  (see  Fig.  106,  I). 
They  might  be  mistaken  by  the  inexperienced  for  red 
blood-corpuscles,  fat-droplets,  or  the  spheric  crystals  of 
calcium  oxalate,  but  are  distinguished  by  the  facts  that 
they  are  not  of  uniform  size;  that  they  tend  to  adhere  in 
short  chains;  that  small  buds  may  often  be  seen  ad- 
hering to  the  larger  cells;  and  that  they  do  not  give  the 
hemoglobin  test,  are  not  stained  by  osmic  acid  or  Sudan, 
but  are  colored  brown  by  Lugol's  solution,  and  are  in- 
soluble in  acids  and  alkalis.     Yeast-cells  multiply  rapidly 


172 


THE   URINE 


in  diabetic  urine,  and  may  reach  the  bladder  and  multi- 
ply there. 

Mold  fungi  (Fig.  62)  are  characterized  by  refractive, 
jointed,  or  branched  rods  (hyphae),  often  arranged  in  a 
network,  and  by  highly  refractive,  spheric  or  ovoid  spores. 


Fig.  61. — Extraneous  matters  found  in  urine:  a.  Flax-fibers;  b,  cotton-fibers;  c.  feathers; 
d,  hairs;  e,  potato-starch;  /,  rice-starch  granules;  g,  wheat-starch;  h,  air-bubbles;  », 
muscular  tissue;  k,  vegetable  tissue;  /,  oil-globules. 


They  are  common  in  urine  which  has  stood  exposed  to 
the  air. 

Fibers  of  wool,  cotton,  linen,  or  silk,  derived  from 
towels,  the  clothing  of  the  patient,  or  the  dust  in  the  air, 
are  present  in  almost  every  urine.  Fat-droplets  are  most 
frequently  derived  from  unclean  bottles  or  oiled  cathe- 


THE    URINE   IN   DISEASE  1 73 

ters.  Starch-granules  may  reach  the  urine  from  towels, 
the  clothing,  or  dusting-powders.  They  are  recognized 
by  their  concentric  striations  and  their  blue  color  with 
iodin  solution.  Lycopodium  granules  (Fig.  5)  may  also 
reach  the  urine  from  dusting-powders.  They  might  be 
mistaken  for  the  ova  of  parasites.  Bubbles  of  air  are 
often  confusing  to  beginners,  but  are  easily  recognized 


Fig.  62. — Aspergill»is  from  urine  (Boston). 

after  once  being  seen.  Scratches  and  flaws  in  the  glass 
of  slide  or  cover  are  likewise  a  common  source  of  con- 
fusion to  beginners. 

IV.  THE  XJRINE  IN  DISEASE 

In  this  section  the  characteristics  of  the  urine  in  those 
diseases  which  produce  distinctive  urinary  changes  will 
be  briefly  reviewed. 

I.  Renal  Hyperemia. — Active  hyperemia  is  usually  an 
early  stage  of  acute  nephritis,  but  may  occur  independ- 
ently as  a  result  of  temporary  irritation.  The  urine  is 
generally  decreased  in  quantity,  highly  colored,  and 
strongly  acid.  Albumin  is  always  present — usually  in 
traces  only,  but  sometimes  in  considerable  amount  for  a 


174 


THE    URINE 


day  or  two.     The  sediment  contains  a  few  hyaline  and 
finely  granular  casts  and  an  occasional  red  blood-cell. 


Fig.  63. — Sediment  from  acute  hemorrhagic  nephritis:  Red  hloo.l-corpuscles;  leukocytes; 
renal  cells  not  fattily  degenerated;  epithelial  and  blood-casts  (Jakob). 


Fig.  64. — Sediment  from  chronic  parenchymatous  nephritis:  Hyaline  (with  cells 
attached),  waxy,  brown  granular,  fatty,  and  epithelial  casts;  fattily  degenerated  renal  cells, 
and  a  few  white  and  red  blood-corpuscles  (Jakob). 

In  very  severe  hyperemia  the  urine  approaches  that  of 
acute  nephritis. 

Passive  hyperemia  occurs  most  commonly  in  diseases  of 


THE   URINE  IN  DISEASE  175 

the  heart  and  liver  and  in  pregnancy.  The  quantity  of 
urine  is  somewhat  low  and  the  color  high,  except  in 
pregnancy.  Albumin  is  present  in  small  amount  only. 
The  sediment  contains  a  very  few  hyaHne  or  finely 
granular  casts.  In  pregnancy  the  amount  of  albumin 
should  be  carefully  watched,  as  any  considerable  quan- 
tity, and  especially  a  rapid  increase,  strongly  suggests 
approaching  eclampsia. 

2.  Nephritis. — The  various  degenerative  and  inflam- 
matory conditions  grouped  under  the  name  of  nephritis 
have  certain  features  in  common.  The  urine  in  all 
cases  contains  albumin  and  tube-casts,  and  in  all  well- 
marked  cases  shows  a  decrease  of  normal  solids,  especially 
of  urea  and  the  chlorids.  In  chronic  nephritis,  especially 
of  the  interstitial  type,  there  may  be  remissions  during 
which  the  urine  is  practically  normal.  The  character- 
istics of  the  different  forms  are  well  shown  in  the  table 
on  page  176,  modified  from  Hill. 

3.  Renal  Tuberculosis. — The  urine  is  pale,  usually 
cloudy.  The  quantity  may  not  be  affected,  but  is  apt  to 
be  increased.  In  early  cases  the  reaction  is  faintly  acid 
and  there  are  traces  of  albumin  and  a  few  renal  cells. 
In  advanced  cases  the  urine  is  alkaline,  has  an  offensive 
odor,  and  is  irritating  to  the  bladder.  Albumin  in  vary- 
ing amounts  is  always  present.  Pus  is  nearly  always 
present,  though  frequently  not  abundant.  It  is  generally 
intimately  mixed  with  the  urine,  and  does  not  settle  so 
quickly  as  the  pus  of  cystitis.  Casts,  though  present,  are 
rarely  abundant,  and  are  obscured  by  the  pus.  Small 
amounts  of  blood  are  common.  Tubercle  bacilli  are 
nearly  always  present,  although  animal  inoculation  may 
be  necessary  to  detect  them. 


176 


THE    URINE 


(—1 

h 

Oh 

s 

w 

l-H 

w 


e  "O  -ii  •- 

o  ^  ^ 

—  pj 

-_  c 

O    rt    ii    i- 

•3OJ 
~    C  ">     J 

o  rt       5  2 

--     -^B'c 
::  >,  i)  3  -u: 

3-c  ^      _ 


r-i  ^      vj      VJ      vj   ' 

g  5  o  i>  c 

^'  =*  S  5 .2 

^-  S   3  .S    fc 

C  .    ^  rf   rt. 


2^§§ 


bc  i:  3 

::::  bc  ^i 


"o 


p  to 

u 
rt 


;  v«  >4-.  o  c  5 


c  5  ,^  3 


rt  2  "5  rt 


•n  3.2 

^-       r/^        ^ 

chlo 
lin: 
cact 

0      i5       ^ 

11^:2 

a  and 
albun 
nt.    R 

■§£2-^ 

rt  rt  ^c 
rt   ci;    3.2 

f^x:  0 

00        -Z! 

J:;  u  u 

t-   tc  . .  u 

►^3    1. 

=^  rt.S  2 

^  a. 

•J  £p^ 

-^1=.^ 

B4i-° 

-=  c      -^ 

^•t;  rt  0 

.ao'-c  rt 

^   rt  J-   « 

.5    .•    >-    bC 

>~.'C        0 

=   rt        J 

dim 
reatl} 

be 
•cific 

0 

usua 
jlor  V 
and 
vity, 

>,tC;^^ 

q 

>.'"-  i)  ^ 

•S  bScT 

" 

•z:       rt  60 

c   a  C 

_c 

rt   -        tc     . 

rt   >         >. 

often 
dark; 
smok 

0 
q 

a-         Qu 

ge     inishi 

often 

Speci; 

1.020, 

n' 

(J 

*-I3 

c 

:s 

2  «•_ 

a. 

S.ti  >» 

u 

^-  w 

c 

o-C  C 

u 

•sg-:2 

3 

p  c^ 

^ 

Chr 
tous 
white 

c    C    CJ 


s  X  &  s 

P  O    O    T 

.£0  •"    g 

"^  .£  Ta       3 
^  =«.S   u  c  c 

-2  ^      ^  •"  .2 
•r       c      .B  u 

-I  s  2  c  2^ 

"if  a.ii  fc 

rt  T  -  bO 

rt^-3        -^5 

•^      3  ?^  .^ 

•S  <  i:  ^  .3 

i  rt  ^2 

—  -^  -r  o 
-c  jC-      o 

-« .5  — 

£  -^  t  ■> 

-      Ui 


.._»  «     Ui 


^  ^u 


rt 


0_bC  ^  O 


C 


i^ 


-8-1 


60 


c/: 


^  £ 


O    rt  •= 

(D    .  r: 

u    to    rt 

c   c  ^ 
rt  rt  bO 


>.  -r 


a  a,> 


s  cj«3  00 


C: 


bC 

u 

£."2 


THE   URINE   IN  DISEASE 


177 


4.  Renal  Calculus. — The  urine  is  usually  somewhat 
concentrated,  with  high  color  and  strongly  acid  reaction. 
Small  amounts  of  albumin  and  a  few  casts  may  be  pres- 
ent as  a  result  of  kidney  irritation.  Blood  is  frequently 
present,  especially  in  the  daytime  and  after  severe  ex- 
ercise. Crystals  of  the  substance  composing  the  cal- 
culus— uric  acid,  calcium  oxalate,  cystin — may  often 
be  found.    The  presence  of  a  calculus  generally  produces 


Fig.  65. — Sediment  from  calculous  pyelitis:  Numerous  pus-corpuscles,  red  blood-cor- 
puscles, and  caudate  and  irregular  epithelial  cells;  a  combination  of  hyaline  and  pus- 
casts,  and  a  few  uric-acid  crystals  (Jakob). 


pyelitis,  and  variable  amounts  of  pus  then  appear,  the 
urine  remaining  acid  in  reaction. 

5.  Pyelitis. — In  pyelitis  the  urine  is  slightly  acid,  and 
contains  a  small  or  moderate  amount  of  pus,  together 
with  many  spindle  and  caudate  epithelial  cells.  Pus- 
" casts  may  appear  if  the  process  extends  up  into  the  kid- 
ney tubules  (see  Fig.  65).  Albumin  is  always  present, 
and  its  amount,  in  proportion  to  the  amount  of  pus,  is 
decidedly  greater  than  is  found  in  cystitis.  This  fact  is 
12 


178 


THE   URINE 


of  much  value  in  differential  diagnosis.  Even  when  pus 
is  scanty,  albumin  is  rarely  under  0.15  per  cent.,  which 
is  the  maximum  amount  found  in  cystitis  with  abundant 
pus. 

6.  Cystitis.— In  acute  and  subacute  cases  the  urine  is 
acid  and  contains  a  variable  amount  of  pus,  with  many 
epithelial  cells  from  the  bladder — chiefly  large  round, 
pyriform,  and  rounded  squamous  cells.  Red  blood- 
corpuscles  are  often  numerous. 


Fig.  66. — Sediment  from  cystitis  (chronic):  Numerous  pus-corpuscles,  epithelial  celb 
from  the  bladder,  and  bacteria;  a  few  red  blood-corpuscles  and  triple  phosphate  and 
ammonium  urate  crystals  (Jakob). 

In  chronic  cases  the  urine  is  generally  alkaline.  It  is 
pale  and  cloudy  from  the  presence  of  pus,  which  is  abun- 
dant and  settles  readily  into  a  viscid  sediment.  The 
sediment  usually  contains  abundant  amorphous  phos- 
phates and  crystals  of  triple  phosphate  and  ammonium 
urate.  Vesical  epithelium  is  common.  Numerous  bac- 
teria are  always  present  (see  Fig.  66). 

7.  Vesical  Calculus,  Tumors,  and  Tuberculosis. — 
These  conditions  produce  a  chronic  cystitis,  with  its 


THE   URINE   IN   DISEASE  1 79 

characteristic  urine.  Blood,  however,  is  more  frequently 
present  and  more  abundant  than  in  ordinary  cystitis. 
With  neoplasms,  especially,  considerable  hemorrhages 
are  apt  to  occur.  Particles  of  the  tumor  are  sometimes 
passed  with  the  urine.  No  diagnosis  can  be  made  from 
the  presence  of  isolated  tumor  cells.  In  tuberculosis 
tubercle  bacilli  can  generally  be  detected. 

8.  Diabetes  Insipidus. — Characteristic  of  this  disease 
is  the  continued  excretion  of  very  large  quantities  of  pale, 
watery  urine,  containing  neither  albumin  nor  sugar. 
The  specific  gravity  varies  between  i.ooi  and  1.005. 
The  daily  output  of  solids,  especially  urea,  is  increased. 

9.  Diabetes  Mellitus. — The  quantity  of  urine  is  very 
large.  The  color  is  generally  pale,  while  the  specific 
gravity  is  nearly  always  high — 1.030  to  1.050,  very  rarely 
below  1.020.  The  presence  of  glucose  is  the  essential 
feature  of  the  disease.  The  amount  of  glucose  is  often 
very  great,  sometimes  exceeding  8  per  cent.,  while  the 
total  elimination  may  exceed  500  gm.  in  twenty-four 
hours.  It  may  be  absent  temporarily.  Acetone  is  gen- 
erally present  in  advanced  cases.  Diacetic  and  oxy- 
butyric  acids  may  be  present,  and  usually  warrant  an 
unfavorable  prognosis.  Accompanying  the  acidosis  there 
is  a  corresponding  increase  in  amount  of  ammonia. 


CHAPTER  III 

THE  BLOOD 

Preliminary  Considerations. — The  blood  consists  of 
a  fluid  of  complicated  and  variable  composition,  the 
plasma,  in  which  are  suspended  great  numbers  of  micro- 
scopic structures:  viz.,  red  corpuscles,  white  corpuscles, 
blood-platelets,  and  blood-dust. 

Red  corpuscles,  or  erythrocytes,  appear  as  biconcave 
discs,  red  when  viewed  by  reflected  light  or  in  thick  layer, 
and  straw  colored  when  viewed  by  transmitted  light  or 
in  thin  layer.  They  give  the  blood  its  red  color.  They 
are  cells  which  have  been  highly  differentiated  for  the 
purpose  of  carrying  oxygen  from  the  lungs  to  the  tissues. 
This  is  accomplished  by  means  of  an  iron-bearing  pro- 
tein, hemoglobin,  which  they  contain.  In  the  lungs 
hemoglobin  forms  a  loose  combination  with  oxgyen, 
which  it  readily  gives  up  when  it  reaches  the  tissues. 
Normal  erythrocytes  do  not  contain  nuclei.  They  are 
formed  from  preexisting  nucleated  cells  in  the  bone- 
marrow. 

White  corpuscles,  or  leukocytes,  are  less  highly  differ- 
entiated cells.  There  are  several  varieties.  They  all 
contain  nuclei,  and  most  of  them  contain  granules  which 
vary  in  size  and  staining  properties.  They  are  formed 
chiefly  in  the  bone-marrow  and  lymphoid  tissues. 

Blood- platelets,  or  blood- plaques ,  are  colorless  or  slightly 
bluish,  spheric  or  ovoid  bodies,  about  one- third  or  one- 

180 


PRELIMINARY  CONSIDERATIONS  l8l 

half  the  diameter  of  an  erythrocyte.     Their  structure, 
nature,  and  origin  have  not  been  definitely  determined. 

The  blood-dust  of  Miiller  consists  of  fine  granules  which 
have  vibratory  motion.  Little  is  known  of  them.  It  has 
been  suggested  that  they  are  granules  from  disintegrated 
leukocytes. 

The  total  amount  of  blood  is  usually  given  as  one- 
thirteenth  of  the  body  weight,  but  more  recent  investi- 
gations indicate  that  it  averages  about  one-twentieth. 

The  reaction  is  alkaline  to  litmus. 

The  color  is  due  to  the  presence  of  hemoglobin  in  the 
red  corpuscles,  the  difference  between  the  bright  red  of 
arterial  blood  and  the  purplish  red  of  venous  blood  de- 
pending upon  the  relative  proportions  of  oxygen  and 
carbon  dioxid.  The  depth  of  color  depends  upon  the 
amount  of  hemoglobin.  In  very  severe  anemias  the 
blood  may  be  so  pale  as  to  be  designated  as  "  watery." 
The  formation  of  carbon-monoxid-hemoglobin  in  coal- 
gas  poisoning  gives  the  blood  a  bright  cherry-red  color; 
while  formation  of  methemoglobin  in  poisoning  with 
potassium  chlorate  and  certain  other  substances  gives 
a  chocolate  color. 

Coagulation  consists  essentially  in  the  transformation 
of  fibrinogen,  one  of  the  proteins  of  the  blood,  into  fibrin 
by  means  of  a  ferment  derived  from  disintegration  of  the 
leukocytes.  The  presence  of  calcium  salts  is  necessary 
to  the  process.  The  resulting  coagulum  is  made  up  of  a 
meshwork  of  fibrin  fibrils  with  entangled  corpuscles  and 
plaques.  The  clear,  straw-colored  fluid  which  is  left 
after  separation  of  the  coagulum  is  called  blood-serum. 
Normally,  coagulation  takes  place  in  two  to  eight  min- . 
utes  after  the  blood  leaves  the  Vessels.     It  is  frequently 


l82 


THE    BLOOD 


desirable  to  determine  the  coagulation  time.  The 
simplest  method  is  to  place  a  drop  of  blood  upon  a  per- 
fectly clean  slide,  and  to  draw  a  needle  through  it  at  half- 
minute  intervals.  When  the  clot  is  dragged  along  by  the 
needle,  coagulation  has  taken  place.  This  method  is 
probably  sufficient  for  ordinary  clinical  work.  For  very 
accurate  results  the  method  of  Russell  and  Brodie,  as 
modified  by  Boggs,  is  recommended.  The  instrument 
is  shown  in  Fig.  67.  A  drop  of  blood  is  placed  upon  the 
cone,  which  is  then  quickly  inverted  in  the  moist  chamber. 


Fig.  67. — Boggs'  coagulation  instrument:  A,  moist  chamber;  R,  glass  cone;  C,  tube  through 
which  air  is  blown. 


By  means  of  a  rubber  bulb  puffs  of  air  are  blown  against 
the  blood  at  intervals,  while  the  motion  of  the  corpuscles 
is  watched  with  a  low-power  objective.  Coagulation  is 
complete  when  the  red  cells  move  only  en  masse  and 
spring  back  to  their  original  position  when  the  current 
ceases.  Coagulation  is  notably  delayed  in  hemophilia 
and  icterus  and  after  administration  of  citric  acid.  It 
is  hastened  by  administration  of  calcium  salts. 

For  certain  purposes,  especially  in  bacteriologic  and 
opsonic    work,   it  is  desirable  to  prevent    coagulation 


PRELIMINARY   CONSIDERATIONS  1 83 

of  the  blood  that  is  withdrawn.  This  may  be  accom- 
plished by  receiving  it  directly  into  a  solution  of  i  per 
cent,  sodium  citrate  (or  ammonium  oxalate)  and  0.85 
per  cent,  sodium  chlorid.  This  precipitates  the  calcium 
salts  which  are  necessary  to  coagulation. 

For  most  clinical  examinations  only  one  drop  of  blood 
is  required.  This  may  be  obtained  from  the  lobe  of  the 
ear,  the  palmar  surface  of  the  tip  of  the  finger,  or,  in  the 
case  of  infants,  the  plantar  surface  of  the  great  toe. 
In  general,  the  finger  will  be  found  most  convenient. 
With  nervous  children  the  lobe  of  the  ear  is  preferable, 
as  it  prevents  their  seeing  what  is  being  done.  An  ede- 
matous or  congested  part  should  be  avoided.     The  site 


l-'ig.  68. — Daland's  blood-lancet. 

should  be  well  rubbed  with  alcohol  to  remove  dirt  and 
epithelial  debris  and  to  increase  the  amount  of  blood 
in  the  part.  After  allowing  sufficient  time  for  the  circu- 
lation to  equalize,  the  skin  is  punctured  with  a  blood 
lancet  (of  which  there  are  several  patterns  upon  the 
market)  or  some  substitute,  as  a  Hagedorn  needle, 
aspirating  needle,  trocar,  or  a  pen  with  one  of  its  nibs 
broken  off.  Nothing  is  more  unsatisfactory  than  an 
ordinary  sewing-needle.  The  lancet  should  be  cleaned 
with  alcohol  before  and  after  using,  but  need  not  be 
sterilized.  It  must  be  very  sharp.  If  the  puncture  be 
made  with  a.  firm,  quick,  rebounding  stroke,  it  is  practically 
painless.     The  first  drop  of  blood  which  appears  should 


184  THE   BLOOD 

be  wiped  away,  and  the  second  used  for  examination. 
The  blood  should  not  be  pressed  out,  since  this  dilutes 
it  with  serum  from  the  tissues;  but  moderate  pressure 
some  distance  above  the  puncture  is  allowable. 

When  a  larger  amount  of  blood  is  required,  it  may  be 
obtained  with  a  sterile  hypodermic  S}Tinge  from  one  of 
the  veins  at  the  elbow,  as  described  on  p.  245. 

CHnical  study  of  the  blood  may  be  discussed  under  the 
following  heads:  I.  Hemoglobin.  II.  Enumeration  of 
erythrocytes.  III.  Color  index.  IV.  Volume  index. 
V.  Enumeration  of  leukocytes.  VI.  Enumeration  of 
plaques.  VII.  Study  of  stained  blood.  VIII.  Blood 
parasites.  IX.  Serum  reactions.  X.  Tests  for  recog- 
nition of  blood.     XL  Special  blood  pathology. 

I.  HEMOGLOBIN 

Hemoglobin  is  an  iron-bearing  protein.  It  is  found 
only  within  the  red  corpuscles,  and  constitutes  about 
90  per  cent,  of  their  weight.  The  actual  amount  of 
hemoglobin  is  never  estimated  clinically:  it  is  the  rela- 
tion which  the  amount  present  bears  to  the  normal  which 
is  determined.  Thus  the  expression,  "  50  per  cent,  hemo- 
globin," when  used  clinically,  means  that  the  blood  con- 
tains 50  per  cent,  of  the  normal.  Theoretically,  the 
normal  would  be  100  per  cent.,  but  with  the  methods  of 
estimation  in  general  use  the  blood  of  healthy  persons 
ranges  from  85  to  105  per  cent.;  these  figures  may,  there- 
fore, be  taken  as  normal. 

Increase  of  hemoglohin,  or  hyperchromemia,  is  un- 
common, and  is  probably  more  apparent  than  real.  It 
accompanies  an  increase  in  number  of  erythrocytes,  and 
may  be  noted  in  change  of  residence  from  a  lower  to  a 


HEMOGLOBIN  1 85 

higher  altitude ;  in  poorly  compensated  heart  disease  with 
cyanosis;  in  concentration  of  the  blood  from  any  cause,  as 
the  severe  diarrhea  of  cholera,  and  in  "  idiopathic 
polycythemia." 

Decrease  of  hemoglobin,  or  oligochromemia,  is  very 
common  and  important.  It  is  the  most  striking  feature 
of  the  secondary  anemias  (p.  277).  Here  the  hemo- 
globin loss  may  be  slight  or  very  great.  In  mild  cases  a 
slight  decrease  of  hemoglobin  is  the  only  blood  change 
noted.  In  very  severe  cases,  especially  in  repeated 
hemorrhages,  malignant  disease,  and  infection  by  the 
hookworm  and  Dibothriocephalus  latus,  hemoglobin 
may  fall  to  15  per  cent.  Hemoglobin  is  always  dimin- 
ished, and  usually  very  greatly,  in  chlorosis,  pernicious 
anemia,  and  leukemia. 

Estimation  of  hemoglobin  is  less  tedious  and  usually 
more  helpful  than  a  red  corpuscle  count.  It  offers 
the  simplest  and  most  certain  means  of  detecting  the 
existence  and  degree  of  anemia,  and  of  judging  the  effect 
of  treatment  in  anemic  conditions.  Pallor,  observed 
clinically,  does  not  always  denote  anemia. 

There  are  many  methods,  but  none  is  entirely  satis- 
factory. Those  which  are  most  widely  used  are  here 
described. 

(i)  Von  Fleischl  Method. — ^The  apparatus  consists  of  a 
stand  somewhat  like  the  base  and  stage  of  a  microscope 
(Fig.  69) .  Under  the  stage  is  a  movable  bar  of  colored  glass, 
shading  from  pale  pink  at  one  end  to  deep  red  at  the  other. 
The  frame  in  which  this  bar  is  held  is  marked  with  a  scale  of 
hemoglobin  percentages  corresponding  to  the  different  shades 
of  red.  By  means  of  a  rack  and  pinion,  the  color-bar  can  be 
moved  from  end  to  end  beneath  a  round  opening  in  the  center 


i86 


THE  BLOOD 


of  the  stage.  A  small  metal  cylinder,  which  has  a  glass  bot- 
tom and  which  is  divided  vertically  into  two  equal  compart- 
ments, can  be  placed  over  the  opening  in  the  stage  so  that  one 
of  its  compartments  lies  directly  over  the  color-bar.  Accom- 
})anying  the  instrument  are  a  number  of  short  capillary  tubes 
in  metal  handles. 

Having  punctured  the  finger-tip  or  lobe  of  the  ear,  as  al- 
ready described,  wipe  oflF  the  first  drop  of  blood,  and  from  the 


Fig.  69. — Von  Fleischl's  hcmoglobinomcter:  a,  Stand;  h.  narrow  wedge-shaped  piece 
of  colored  glass  fitted  into  a  frame  ic),  which  passes  under  the  chamber;  d,  hollow  metal 
cylinder,  divided  into  two  compartments,  which  holds  the  blood  and  water;  e.  plaster-of- 
Paris  plate  from  which  the  light  is  reflected  through  the  chamber;  /,  screw  by  which  the 
frame  containing  the  graduated  colored  glass  is  moved;  g,  capillary  tube  to  collect  the 
blood;  h,  pipet  for  adding  the  water;  «,  opening  through  which  may  be  seen  the  scale 
(ixlicating  jiercentage  of  hemoglobin. 


second  fill  one  of  the  capillary  tubes.  Hold  the  tube  hori- 
zontally, and  touch  its  tip  to  the  drop  of  blood,  which  will 
readily  flow  into  it  if  it  be  clean  and  dry.  Avoid  getting  any 
blood  upon  its  outer  surface.  With  a  medicine-dropper,  rinse 
the  blood  from  the  tube  into  one  of  the  compartments  of  the 
cylinder,  using  distilled  water,  and  mix  well.     Fill  both  com- 


HEMOGLOBIN  1 87 

partments  level  full  with  distilled  water,  and  place  the  cylin- 
der over  the  opening  in  the  stage,  so  that  the  compartment 
which  contains  only  water  lies  directly  over  the  bar  of  colored 
glass.  If  there  are  any  clots  in  the  hemoglobin  compart- 
ment, clean  the  instrument  and  begin  again. 

In  a  dark  room,  with  the  light  from  a  candle  reflected  up 
through  the  cylinder,  move  the  color-bar  along  with  a  jerking 
motion  until  both  compartments  have  the  same  depth  of  color. 
The  number  upon  the  scale  corresponding  to  the  portion  of  the 
color-bar  which  is  now  under  the  cylinder  gives  the  percentage 
of  hemoglobin.  While  comparing  the  two  colors,  place  the 
instrument  so  that  they  will  fall  upon  the  right  and  left  halves 
of  the  retina,  rather  than  upon  the  upper  and  lower  halves; 
and  protect  the  eye  from  the  light  with  a  cylinder  of  paper  or 
pasteboard.  After  use,  clean  the  metal  cylinder  with  water, 
and  wash  the  capillary  tube  with  water,  alcohol,  and  ether, 
successively.  Results  with  this  instrument  are  accurate  to 
within  about  5  per  cent. 

A  recent  modification  of  the  von  Fleischl  apparatus  by 
Miescher  gives  an  error  which  need  not  exceed  i  per  cent. 
It  is,  however,  better  adapted  to  laboratory  use  than  to  the 
needs  of  the  practitioner. 

(2)  The  Sahli  hemoglobinometer  (Fig.  70)  is  an  improved 
form  of  the  well-known  Gowers  instrument.  It  consists  of  a 
hermetically  sealed  comparison  tube  containing  a  i  per  cent, 
solution  of  acid  hematin,  a  graduated  test-tube  of  the  same 
diameter,  and  a  pipet  of  20-c.mm.  capacity.  The  two  tubes 
are  held  in  a  black  frame  with  a  white  ground-glass  back. 

Place  a  few  drops  of  decinormal  hydrochloric  acid  solution 
in  the  graduated  tube.  Obtain  a  drop  of  blood  and  draw  it 
"into  the  pipet  to  the  20  c.mm.  mark.  Wipe  off  the  tip  of  the 
pipet,  blow  its  contents  into  the  hydrochloric  acid  solution  in 
the  tube,  and  rinse  well.  In  a  few  minutes  the  hemoglobin  is 
changed  to  acid  hematin.  Place  the  two  tubes  in  the  com- 
partments of  the  frame,  and  dilute  the  fluid  with  water  drop 


i88 


THE    BLOOD 


by  drop,  mixing  after  each  addition,  until  it  has  exactly  the 
same  color  as  the  comparison  tube.  The  graduation  corre- 
sponding to  the  surface  of  the  fluid  then  indicates  the  per- 
centage of  hemoglobin.  Decinormal  hydrochloric  acid  solu- 
tion may  be  prepared  with  suflficient  accuracy  for  this  purpose 


Fig.  70. — Sahli's  hemoglobinometer. 


by  adding  15  c.c.  of  the  concentrated  acid  to  985  c.c.  distilled 
water.  A  little  chloroform  should  be  added  as  a  preservative. 
This  method  is  very  satisfactory  in  practice,  and  is  accurate 
to  within  5  per  cent.  The  comparison  tube  is  said  to  keep  its 
color  indefinitely,  but,  unfortunately,  not  all  the  instruments 
upon  the  market  are  well  standardized. 


HEMOGLOBIN  1 89 

(3)  Dare's  hemoglobinometer  (Fig.  71)  differs  from  the 
others  in  using  undiluted  blood.  The  blood  is  allowed  to  flow 
by  capillarity  into  the  slit  between  two  small  plates  of  glass. 
It  is  then  placed  in  the  instrument  and  compared  with  differ- 
ent portions  of  a  circular  disc  of  colored  glass.  The  reading 
must  be  made  quickly,  before  clotting  takes  place.  This 
instrument  is  easy  to  use,  and  is  one  of  the  most  accurate. 


Fig.  71. — Dare's  hemoglobinometer. 

(4)  Hammerschlag  Method. — This  is  an  indirect  method 
which  depends  upon  the  fact  that  the  percentage  of  hemo- 
globin varies  directly  with  the  specific  gravity  of  the  blood. 
It  yields  fairly  accurate  results  except  in  leukemia,  where  the 
large  number  of  leukocytes  disturbs  the  relation,  and  in 
dropsical  conditions. 

Mix  chloroform  and  benzol  in  a  urinometer  tube,  so  that 
the  specific  gravity  of  the  mixture  is  near  the  probable  specific 
gravity  of  the  blood.  Add  a  drop  of  blood  by  means  of  a 
pipet  of  small  caliber.  A  pipet  hke  that  shown  in  Fig.  161,  A 
will  be  found  satisfactory.  If  the  drop  floats  near  the  surface, 
add  a  little  benzol ;  if  it  sinks  to  the  bottom,  add  a  little  chloro- 
form.   When  it  remains  stationary  near  the  middle,  the  mix- 


IQO 


THE    BLOOD 


ture  has  the  same  specific  gravity  as  the  blood.  Take  the 
specific  gravity  with  a  urinometer,  and  obtain  the  correspond- 
ing percentage  of  hemoglobin  from  the  following  table: 


Gravity. 


Hemoglobin 
Per  Cent. 


Specific 
Gravity. 


Hemoglobin 
Per  Cent- 


1.033-1.035 25-30        1.048-1.050 s5-^5 


1.035-1.038 30-35 

1.038-1.040 35-40 

1.040-1.045 40-45 

1.045-1.048 45-55 


1. 050-1.053 65-70 

1.053-1.055 70-75 

1.055-1.057 75-85 

1.057-1.060 85-95 


For  accurate  results  with  this  method,  care  and  patience 
are  demanded.     The  following  precautions  must  be  observed: 


Fig.  72. — Tallquist's  bemoglobin  scale. 


(a)  The  two  fluids  must  be  well  mixed  after  each  addition 
of  chloroform  or  benzol.  Close  the  tube  with  the  thumb  and 
invert  several  times.     Should  this  cause  the  drop  of  blood  to 


HEMOGLOBIN  I9I 

break  up  into  very  small  ones,  adjust  the  specific  gravity  as 
accurately  as  possible  with  these,  and  test  it  with  a  fresh  drop. 

(b)  The  drop  of  blood  must  not  be  too  large;  it  must  not 
contain  an  air-bubble,  it  must  not  adhere  to  the  side  of  the 
tube,  and  it  must  not  remain  long  in  the  fluid. 

(c)  The  urinometer  must  be  standardized  for  the  chloro- 
form-benzol mixture.  Most  urinometers  give  a  reading  two 
or  three  degrees  too  high,  owing  to  the  low  surface  tension. 
Make  a  mbcture  such  that  a  drop  of  distilled  water  will  re- 
main suspended  in  it  {i.  e.,  with  a  specific  gravity  of  i.ooo) 
and  correct  the  urinometer  by  this. 

(5)  Tallquist  Method. — The  popular  Tallquist  hemo- 
globinometer  consists  simply  of  a  book  of  small  sheets  of  ab- 
sorbent paper  and  a  carefully  printed  scale  of  colors  (Fig.  72). 

Take  up  a  large  drop  of  blood  with  the  absorbent  paper, 
and  when  the  humid  gloss  is  leaving,  before  the  air  has  dark- 
ened the  hemoglobin,  compare  the  stain  with  the  color 
scale.  The  color  which  it  matches  gives  the  percentage 
of  hemoglobin.  Except  in  practised  hands,  this  method  is 
accurate  only  to  within  10  or  20  per  cent. 

Of  the  methods  given,  the  physician  should  select  the 
one  which  best  meets  his  needs.  With  any  method, 
practice  is  essential  to  accuracy.  The  von  Fleischl  has 
long  been  the  standard  instrument,  but  has  lately  fallen 
into  some  disfavor.  For  accurate  work  the  best  instru- 
ments are  the  von  Fleischl-Miescher  and  the  Dare.  They 
are,  however,  expensive,  and  it  is  doubtful  whether  they 
are  enough  more  accurate  than  the  Sahli  instrument  to 
justify  the  difference  in  cost.  The  latter  is  probably  the 
most  satisfactory  for  the  practitioner,  provided  a  well- 
standardized  color- tube  is  obtained.  The  specific  gravity 
method  is  very  useful  when  special  instruments  are  not 
at  hand.     The  Tallquist  scale  is  so  inexpensive  and  so 


192  THE   BLOOD 

convenient  that  it  should  be  used  by  every  physician  at 
the  bedside  and  in  hurried  office  work ;  but  it  should  not 
supersede  the  more  accurate  methods. 

II.  ENUMERATION  OF  ERYTHROCYTES 

In  health  there  are  about  5,000,000  red  corpuscles  per 
cubic  millimeter  of  blood.  Normal  variations  are  shght. 
The  number  is  generally  a  little  less — about  4,500,000 — in 
women. 

Increase  of  red  corpuscles,  or  polycythemia,  is  unimpor- 
tant. There  is  a  decided  increase  following  change  of 
residence  from  a  lower  to  a  higher  altitude,  averaging 
about  50,000  corpuscles  for  each  1000  feet,  but  frequently 
much  greater.  The  increase,  however,  is  not  permanent. 
In  a  few  months  the  erythrocytes  return  to  nearly  their 
original  number.  Three  views  are  ofTered  in  explanation : 
(a)  Concentration  of  the  blood,  owing  to  increased  evap- 
oration from  the  skin;  (b)  stagnation  of  corpuscles  in  the 
peripheral  vessels  because  of  lowered  blood-pressure; 
(c)  new  formation  of  corpuscles,  this  giving  a  compensa- 
tory increase  of  aeration  surface. 

Pathologically,  polycythemia  is  uncommon.  It  may 
occur  in:  (a)  Concentration  of  the  blood  from  severe 
watery  diarrhea;  (b)  chronic  heart  disease,  especially  the 
congenital  variety,  with  poor  compensation  and  .cyanosis; 
and  (c)  idiopathic  polycythemia,  which  is  considered  to  be 
an  independent  disease,  and  is  characterized  by  cyanosis, 
blood  counts  of  7,000.000  to  10,000,000,  hemoglobin  120 
to  150  per  cent.,  and  a  normal  number  of  leukocytes. 

Decrease  of  red  corpuscles,  or  oligocythemia.  Red 
corpuscles  and  hemoglobin  are  commonly  decreased 
together,  although  usually  not  to  the  same  extent. 


ENUMERATION  OF  ERYTHROCYTES 


195 


Oligocythemia  occurs  in  all  but  the  mildest  symp- 
tomatic anemias.  The  blood-count  varies  from  near  the 
normal  in  moderate  cases  down  to  1,500,000  in  very 
severe  cases.  There  is  always  a  decrease  of  red  cells  in 
chlorosis,  but  it  is  often  slight,  and  is  relatively  less  than 
the  decrease  of  hemoglobin.  Leukemia  gives  a  decided 
oligocythemia,  the  average  count  being  about  3,000,000. 
The  greatest  loss  of  red  cells  occurs  in  pernicious  anemia, 
where  counts  below  1,000,000  are  not  uncommon. 


OlOOmm 

H 

73. — Thoma-Zeiss  heraocytometer:  a.  Slide  used  in  counting;  h,  sectional  view; 
d,  red  pipet;  e,  white  pipet. 

The  most  widely  used  and  most  satisfactory  instru- 
ment for  counting  the  corpuscles  is  that  of  Thoma-Zeiss, 
The  hematocrit  is  not  to  be  recommended  for  accuracy, 
since  in  anemia,  where  blood-counts  are  most  important, 
the  red  cells  vary  greatly  in  size  and  probably  also  in 
elasticity.  The  hematocrit  is,  however,  useful  in  de- 
termining the  relative  volume  of  corpuscles  and  plasma 
(Volume  Index,  p.  200),  and  seems  to  be  gaining  in  favor. 

13 


194 


THE   BLOOD 


The  Thoma-Zeiss  instrument  consists  of  two  pipets 
for  diluting  the  blood  and  a  counting  chamber  (Fig.  73). 
The  counting  chamber  is  a  glass  slide  with  a  square  platform 
in  the  middle.  In  the  center  of  the  platform  is  a  circular 
opening,  in  which  is  set  a  small  circular  disc  in  such  a  manner 
that  it  is  surrounded  by  a  "  ditch,"  and  that  its  surface  is 


Fig.  74. — Ordinary  ruling   of   counting  chamber,  showing  red  corpuscles  in  left 


upper 


exactly  one-tenth  of  a  millimeter  below  the  surface  of  the 
square  platform.  Upon  this  disc  is  ruled  a  square  millimeter, 
subdivided  into  400  small  squares.  Each  fifth  row  of  small 
squares  has  double  rulings  for  convenience  in  counting  (Fig. 
74).  A  thick  cover-glass,  ground  perfectly  plane,  accompa- 
nies the  counting  chamber.  Ordinary  cover-glasses  are  of 
uneven  surface,  and  should  not  be  used  with  this  instrument. 


ENUMERATION   OF   ERYTHROCYTES  1 95 

It  is  evident  that,  when  the  cover-glass  is  in  place  upon 
the  platform,  there  is  a  space  exactly  one-tenth  of  a  millimeter 
thick  between  it  and  the  disc;  and  that,  therefore,  the  square 
millimeter  ruled  upon  the  disc  forms  the  base  of  a  space 
holding  exactly  one-tenth  of  a  cubic  millimeter. 

Technic. — To  count  the  red  corpuscles,  use  the  pipet  with 
loi  engraved  above  the  bulb.  It  must  be  clean  and  dry. 
Obtain  a  drop  of  blood  as  already  described.     Suck  blood 


Fig.  75. — Method  of  drawing  blood  into  the  pipet  (Boston), 

into  the  pipet  to  the  mark  0.5  or  i.  Should  the  blood  go 
beyond  the  mark,  draw  it  back  by  touching  the  tip  of  the  pipet 
to  a  moistened  handkerchief.  Quickly  wipe  off  the  blood 
adhering  to  the  tip,  plunge  it  into  the  diluting  fluid,  and  suck 
the  fluid  up  to  the  mark  loi,  slightly  rotating  the  pipet 
meanwhile.  This  dilutes  the  blood  i  :  200  or  i  :  100,  accord- 
ing to  the  amount  of  blood  taken.  Except  in  cases  of  severe 
anemia,  a  dilution  of  i  :  200  is  preferable.     Close  the  ends 


196  THE    BLOOD 

of  the  pipet  with  the  fingers,  and  shake  vigorously  until  the 
blood  and  diluting  fluid  are  well  mixed. 

When  it  is  not  convenient  to  count  the  corpuscles  at  once, 
place  a  heavy  rubber  band  around  the  pipet  so  as  to  close 
the  ends,  inserting  a  small  piece  of  rubber-cloth  or  other 
tough,  non-absorbent  material,  if  necessary,  to  prevent  the  tip 
from  punching  through  the  rubber.  It  may  be  kept  thus  for 
twenty-four  hours  or  longer. 

When  ready  to  make  the  count,  clean  the  counting 
chamber  and  cover-glass,  and  place  a  sheet  of  paper  over 
them  to  keep  off  dust.  IVIix  the  fluid  thoroughly  by  shak- 
ing; blow  two  or  three  drops  from  the  pipet,  wipe  of!  its 
tip,  and  then  place  a  small  drop  (the  proper  size  can  be 
learned  only  by  experience)  upon  the  disc  of  the  counting 
chamber.  Adjust  the  cover  immediately.  Hold  it  by  diag- 
onal corners  above  the  drop  of  fluid  so  that  a  third  corner 
touches  the  slide  and  rests  upon  the  edge  of  the  platform. 
Place  a  finger  upon  this  corner,  and,  by  raising  the  finger, 
allow  the  cover  to  fall  quickly  into  place.  If  the  cover  be 
properly  adjusted,  faint  concentric  lines  of  the  prismatic 
colors — Newton's  rings — can  be  seen  between  it  and  the  plat- 
form when  the  slide  is  viewed  obliquely.  They  indicate  that 
the  two  surfaces  are  in  close  apposition.  If  they  do  not  ap- 
pear at  once,  slight  pressure  upon  the  cover  may  bring  them 
out.  Failure  to  obtain  them  is  usually  due  to  dirty  slide  or 
cover — both  must  be  perfectly  clean  and  free  from  dust. 
The  drop  placed  upon  the  disc  must  be  of  such  size  that,  when 
the  cover  is  adjusted,  it  nearly  or  quite  covers  the  disc,  and 
that  none  of  it  runs  over  into  the  "  ditch."  There  should 
be  no  bubbles  upon  the  ruled  area. 

Allow  the  corpuscles  to  settle  for  a  few  minutes,  and  then 
examine  with  a  low  power  to  see  that  they  are  evenly  dis- 
tributed. If  they  are  not  evenly  distributed  over  the  whole  disc. 
the  counting  chamber  must  be  cleaned  and  a  new  drop  placed 
in  it. 


ENUMERATION   OF   ERYTHROCYTES 


197 


Probably  the  most  satisfactory  objective  for  counting  is  the 
special  4  mm.  with  long  working  distance.  To  understand 
the  principle  of  counting,  it  is  necessary  to  remember  that 
the  large  square  (400  small  squares)  represents  a  capacity 
of  one-tenth  of  a  cubic  millimeter.  Find  the  number  of 
corpuscles  in  the  large   square,  multiply  by  10  to  find  the 


Fig.  76. — Appearance  of  microscopic  field  in  counting  red  corpuscles.    The  arrow  indicates 
the  squares  to  be  counted. 


number  in  i  c.mm.  of  the  diluted  blood,  and  finally,  by  the 
dilution,  to  find  the  number  in  i  c.mm.  of  undiluted  blood. 
.  Instead  of  actually  counting  all  the  corpuscles,  it  is  customary 
to  count  those  in  only  a  limited  number  of  small  squares, 
and  from  this  to  calculate  the  number  in  the  large  square. 
Nearly  every  worker  has  his  own  method  of  doing  this. 
The  essential  thing  is  to  adopt  a  method  and  adhere  to  it. 


198  THE   BLOOD 

In  practice  a  convenient  procedure  is  as  follows:  With  a 
dilution  of  i  :  200,  count  the  cells  in  80  small  squares,  and  to 
the  sum  add  4  ciphers;  with  dilution  of  i  :  100,  count  40  small 
squares  and  add  4  ciphers.  Thus,  if  with  i  :  200  dilution,  450 
corpuscles  were  counted  in  80  squares,  the  total  count  would 
be  4,500,000  per  c.mm.  This  method  is  sufficiently  accurate 
for  all  clinical  purposes,  provided  the  corpuscles  are  evenly 
distributed  and  three  drops  from  the  pipet  be  counted.  It  is 
convenient  to  count  a  block  of  20  small  squares,  as  indicated 
in  Fig.  76,  in  each  corner  of  the  large  square.  Four  columns 
of  5  squares  each  are  counted.  The  double  rulings  show  when 
the  bottom  of  a  column  has  been  reached  and  also  indicate 
the  fourth  column.  In  the  writer's  opinion  it  is  easier  to 
count  in  vertical  than  horizontal  rows.  If  distribution  be 
even,  the  difference  between  the  number  of  cells  in  any  two 
such  blocks  should  not  exceed  twenty.  In  order  to  avoid 
confusion  in  counting  cells  which  lie  upon  the  border-lines, 
the  following  rule  is  generally  adopted:  Corpuscles  which 
touch  the  upper  and  left  sides  should  be  counted  as  if  within  the 
squares,  those  touching  the  lower  and  right  sides,  as  outside;  and 
vice  versd.. 

Diluting  Fluids. — The  most  widely  used  are  Hayem's  and 
Toisson's.  Both  of  these  have  high  specific  gravities,  so  that, 
when  well  mixed,  the  corpuscles  do  not  separate  quickly. 
Toisson's  fluid  is  probably  the  better  for  beginners,  because  it 
is  colored  and  can  easily  be  seen  as  it  is  drawn  into  the  pipet. 
It  stains  the  nuclei  of  leukocytes  blue,  but  this  is  no  real  ad- 
vantage.    It  must  be  filtered  frequently. 

Hayem's  Fluid.  Toisson's  Fturo. 

Mercuric  chlorid 0.5      Methyl-violet,  5  B 0.025 

Sodium  sulphate    5.0      Sodium  chlorid i.ooo 

Sodium  chlorid i.o      Sodium  sulphate 8.000 

Distilled  water 200.0      Glycerin 30.000 

Distilled  water 160.000 


ENUMERATION  OF  ERYTHROCYTES  1 99 

Sources  of  Error. — The  most  common  sources  of  error  in 
making  a  blood  count  are: 

(a)  Inaccurate  dilution,  either  from  faulty  technic  or 
inaccurately  graduated  pipets.  The  instruments  made  by 
Zeiss  can  be  relied  upon. 

(b)  Too  slow  manipulation,  allowing  a  little  of  the  blood  to 
coagulate  and  remain  in  the  capillary  portion  of  the  pipet, 

(c)  Inaccuracy  in  depth  of  counting  chamber,  which  some- 
times results  from  softening  of  the  cement  by  alcohol  or  heat. 
The  slide  should  not  be  cleaned  with  alcohol  nor  left  to  lie 
in  the  warm  sunshine. 

(d)  Uneven  distribution  of  the  corpuscles.  This  results 
when  the  blood  is  not  thoroughly  mixed  with  the  diluting 
fluid,  or  when  the  cover-glass  is  not  applied  soon  enough  after 
the  drop  is  placed  upon  the  disc. 

Cleaning  the  Instrument. — The  instrument  should  be 
cleaned  immediately  after  using,  and  the  counting  chamber 
and  cover  must  be  cleaned  again  just  before  use. 

Draw  through  the  pipet,  successively,  water,  alcohol,  ether, 
and  air.  This  can  be  done  with  the  mouth,  but  it  is  much 
better  to  use  a  rubber  bulb  or  suction  filter  pump.  When  the 
mouth  is  used,  the  moisture  of  the  breath  will  condense  upon 
the  interior  of  the  pipet  unless  the  fluids  be  shaken  and  not 
blown  out.  If  blood  has  coagulated  in  the  pipet — which  hap- 
pens when  the  work  is  done  too  slowly — dislodge  the  clot  with 
a  horsehair,  and  clean  with  strong  sulphuric  acid,  or  let  the 
pipet  stand  over  night  in  a  test-tube  of  the  acid.  Even  if  the 
pipet  does  not  become  clogged,  it  should  be  occasionally 
cleaned  in  this  way.  When  the  etched  graduations  on  the 
pipets  become  dim,  they  can  be  renewed  by  rubbing  with  a 
grease  pencil. 

Wash  the  counting-chamber  and  the  cover  with  water  and 
dry  them  with  clean  soft  linen.  Alcohol  may  be  used  to  clean 
the  latter,  but  never  the  former. 


200  THE   BLOOD 

III.  COLOR  INDEX 

This  is  an  expression  which  indicates  the  amount  of 
hemoglobin  in  each  red  corpuscle  compared  with  the 
normal  amount.  For  example,  a  color  index  of  i.o 
indicates  that  each  corpuscle  contains  the  normal  amount 
of  hemoglobin;  of  0.5,  that  each  contains  one-half  the 
normal. 

The  color  index  is  most  significant  in  chlorosis  and 
pernicious  anemia.  In  the  former  it  is  usually  much 
decreased;  in  the  latter,  generally  much  increased.  In 
symptomatic  anemia  it  is  generally  moderately  dimin- 
ished. 

To  obtain  the  color  index,  divide  the  percentage  of  hemo- 
globin by  the  percentage  of  corpuscles.  The  percentage  of 
corpuscles  is  found  by  multiplying  the  first  two  figures  of  the 
red  corpuscle  count  by  two.  This  simple  method  holds  good 
for  all  counts  of  1,000,000  or  more.  Thus,  a  count  of  2,500,000 
is  50  per  cent,  of  the  normal.  If,  then,  the  hemoglobin  has 
been  estimated  at  40  per  cent.,  divide  40  (the  percentage  of 
hemoglobin)  by  50  (the  percentage  of  corpuscles).  This 
gives  i,  or  0.8,  as  the  color  index. 

IV.  VOLUME  INDEX 

The  term  "  volume  index  "  was  introduced  by  Capps 
to  express  the  average  size  of  the  red  cells  of  an  individual 
compared  with  their  normal  size.  It  is  the  quotient 
obtained  by  dividing  the  volume  of  red  corpuscles  (ex- 
pressed in  percentage  of  the  normal)  by  the  number  of 
red  corpuscles,  also  expressed  in  percentage  of  the  nor- 
mal. 

The  volume  index  more  or  less  closely  parallels  the 
color  index,  and  variations  have  much  the  same  sig- 


^m^  rr-r  rr-  ^^^ 


VOLUME 

r.  u  ■,  r  r^  '  J    '^1  /^  I 

mficance.    The  following  are  avetages-  6^  ttr^  examma- 

tions  reported  by  Larrabee  in  the  Journal  of  Medical       ^ 

Research: 

Red  corpuscles  Hemoglobin  per  _  ,                , ,  , 

,  .  ^   u    c  Li-  Color             Volume 

per  cubic  cent  by  Sahli  .    . 

.....  .    .           .  mdez.             mdex. 

millimeter.  instrument. 

Normal  males 5,267,250  103.0  0.98  1.007 

Normal  females 4,968,667  106.0  1.06  i.ooi 

Primary  pernicious  anemia .  1,712,166  50.0  1.47  1.270 

Secondary  anemia 3, 737, 160  61.0  0.81  0.790 

Chlorosis 3,205,000  34.5  0.55  0.695 

Method. — The  red  cells  are  counted  and  the  percentage  of 
red  cells  calculated  as  for  the  color  index. 

The  volume  percentage  is  obtained  with  the  hematocrit 
as  follows:  Fill  the  hematocrit  tubes  (Fig.  77)  with  blood,  and 
before  coagulation  takes  place  insert  them  in  the  frame  and 
centrifugalize  for  three  minutes  at  about  8000  to  10,000 
revolutions  a  minute.  The  red  cells  collect  at  the  bottom 
and,  normally,  make  up  one-half  of  the  total  column  of  blood. 


{  "10' •■£;^''j^Q-;;j^';rj^<'^^''^' q'q' g^ 


Fig.  77. — Daland  hematocrit  for  use  with  the  centrifuge. 

Multiply  the  height  of  the  layer  of  red  cells  (as  indicated  by 
the  graduations  upon  the  side  of  the  tube)  by  2  to  obtain  the 
volume  percentage.  When  the  examination  cannot  be  made 
immediately  after  the  blood  is  obtained,  the  method  of 
Larrabee  is  available.  This  consists  in  mixing  a  trace  of  so- 
dium oxalate  with  a  few  drops  of  blood  to  prevent  coagulation, 
drawing  this  mixture  into  a  tube  of  about  2-mm.  caliber  and 
waiting  until  sedimentation  is  complete — usually  about  three 
days.    The  height  of  the  column  is  then  measured  with  a 


202  XMK    BLOOD 

miUimetpr  saiJe  and  tlie  percentage  relation  to  the  normal 
calculated. 

After  the  volume  of  the  red  cells  and  the  red  corpuscle 
count  are  thus  expressed  in  percentages,  divide  the  former 
by  the  latter  to  find  the  volume  index.  Example:  Suppose 
the  volume  percentage  is  80  (the  reds  reaching  to  mark  40  on 
hematocrit  tube)  and  that  the  red  count  is  50  per  cent,  of 
the  normal  (2,500,000  per  c.mm.),  then  |^  or  1.6  is  the  vol- 
ume index. 

V.  ENUMERATION  OF  LEUKOCYTES 

The  normal  number  of  leukocytes  varies  from  5000  to 
10,000  per  cubic  millimeter  of  blood.  The  number  is 
larger  in  robust  individuals  than  in  poorly  nourished 
ones,  and  if  disease  be  excluded,  may  be  taken  as  a 
rough  index  of  the  individual's  nutrition.  Since  it  is 
well  to  have  a  definite  standard,  7500  is  generally  adopted 
as  the  normal  for  the  adult.  With  children  the  number 
is  somewhat  greater,  counts  of  12,000  and  15,000  being 
common  in  healthy  children  under  twelve  years  of  age. 

Decrease  in  Number  of  Leukocytes 

Decrease  in  number  of  leukocytes,  or  leukopenia,  is  not 
important.  It  is  common  in  persons  who  are  poorly 
nourished,  although  not  actually  sick.  The  infectious 
diseases  in  which  leukocytosis  is  absent  (p.  206)  often 
cause  a  slight  decrease  of  leukocytes.  Chlorosis  may 
produce  leukopenia,  as  also  pernicious  anemia,  which 
usually  gives  it  in  contrast  to  the  secondary  anemias, 
which  are  frequently  accompanied  by  leukocytosis. 
Leukocyte  counts  are,  therefore,  of  some  aid  in  the  dififer- 
ential  diagnosis  of  these  conditions. 


enumeration  of  leukocytes  203 

Increase  in  Number  of  Leukocytes 
Increase  in  number  of  leukocytes  is  common  and  of 
great  importance.     It  may  be  considered  under   two 
heads: 

A.  Increase  of  leukocytes  due  to  chemotaxis  and 
stimulation  of  the  blood-making  organs,  or  leukocytosis. 
The  increase  affects  one  or  more  of  the  normal  varieties. 

B.  Increase  of  leukocytes  due  to  leukemia.  Normal 
varieties  are  increased,  but  the  characteristic  feature  is 
the  appearance  of  great  numbers  of  abnormal  cells. 

The  former  may  be  classed  as  a  transient,  the  latter,  as 
a  permanent,  increase. 

A.     Leukocytosis 

This  term  is  variously  used.  By  some  it  is  applied  to 
any  increase  in  number  of  leukocytes;  by  others  it  is 
restricted  to  increase  of  the  polymorphonuclear  neutro- 
philic variety.  As  has  been  indicated,  it  is  here  taken 
to  mean  a  transient  increase  in  number  of  leukocytes, 
that  is,  one  caused  by  chemotaxis  and  stimulation  of  the 
blood-producing  structures,  in  contrast  to  the  permanent 
increase  caused  by  leukemia. 

By  chemotaxis  is  meant  that  property  of  certain  agents 
by  which  they  attract  or  repel  living  cells — positive 
chemotaxis  and  negative  chemotaxis  respectively.  An 
excellent  illustration  is  the  accumulation  of  leukocytes 
at  the  site  of  inflammation,  owing  to  the  positively 
chemotactic  influence  of  bacteria  and  their  products.  A 
great  many  agents  possess  the  power  of  attracting  leuko- 
cytes into  the  general  circulation.  Among  these  are 
many  bacteria  and  certain  organic  and  inorganic  poisons. 

Chemotaxis  alone  will  not  explain  the  continuance  of 


204  THE   BLOOD 

leukocytosis  for  more  than  a  short  time.  It  is  probable 
that  substances  which  are  positively  chemotactic  also 
stimulate  the  blood-producing  organs  to  increased  forma- 
tion of  leukocytes;  and  in  at  least  one  form  of  leukocytosis 
such  stimulation  apparently  plays  the  chief  part. 

As  will  be  seen  later,  there  are  several  varieties  of  leu- 
kocytes in  normal  blood,  and  most  chemotactic  agents 
attract  only  one  variety,  and  either  repel  or  do  not  in- 
fluence the  others.  It  practically  never  happens  that 
all  are  increased  in  the  same  proportion.  The  most 
satisfactory  classification  of  leukocytoses  is,  therefore, 
based  upon  the  type  of  leukocyte  chiefly  affected. 

Theoretically,  there  should  be  a  subdivision  for  each 
variety  of  leukocyte,  e.  g.,  polymorphonuclear  leuko- 
cytosis, lymphocyte  leukocytosis,  eosinophilic  leuko- 
cytosis, large  mononuclear  leukocytosis,  etc.  Practi- 
cally, however,  only  two  of  these,  polymorphonuclear 
leukocytosis  and  lymphocyte  leukocytosis,  need  be  con- 
sidered under  the  head  of  Leukocytosis.  Increase  in 
number  of  the  other  leukocytes  will  be  considered 
when  the  individual  cells  are  described  (pp.  230-243). 
They  are  present  in  the  blood  in  such  small  numbers 
normally  that  even  a  marked  increase  scarcely  afi^ects 
the  total  leukocyte  count;  and,  besides,  substances 
which  attract  them  into  the  circulation  frequently  repel 
the  pol>'morphonuclears,  so  that  the  total  number  of 
leukocytes  may  actually  be  decreased. 

The  polymorphonuclear  neutrophils  are  capable  of 
active  ameboid  motion,  and  are  by  far  the  most  numerous 
of  the  leukocytes.  Ljonphocytes  are  about  one-third 
as  numerous  and  have  little  independent  motion.  As 
one  would,  therefore,  expect,  marked  differences  exist 


ENUMERATION   OF   LEUKOCYTES  205 

between  the  two  types  of  leukocytosis:  polynuclear 
leukocytosis  is  more  or  less  acute,  coming  on  quickly  and 
often  reaching  high  degree;  whereas  lymphocyte  leuko- 
cytosis is  more  chronic,  comes  on  more  slowly,  and  is 
never  so  marked. 

I.  Polymorphonuclear  Neutrophilic  Leukocytosis. — 
Polymorphonuclear  leukocytosis  may  be  either  physi- 
ologic or  pathologic.  A  count  of  20,000  would  be  con- 
sidered a  marked  leukocytosis;  of  30,000,  high;  above 
50,000,  extremely  high. 

(i)  Physiologic  Polymorphonuclear  Leukocytosis. — 
This  is  never  very  marked,  the  count  rarely  exceeding 
15,000  per  cubic  millimeter.  It  occurs:  (a)  In  the  new- 
born; (b)  in  pregnancy;  (c)  during  digestion,  and  (d) 
after  cold  baths.  There  is  moderate  leukocytosis  in  the 
moribund  state:  this  is  commonly  classed  as  physiologic, 
but  is  probably  due  mainly  to  temiinal  infection. 

The  increase  in  these  conditions  is  not  limited  to  the 
polymorphonuclears.  Lymphocytes  are  likewise  in- 
creased in  varying  degrees,  most  markedly  in  the  new- 
bom. 

In  view  of  the  leukocytosis  of  digestion,  the  hour 
at  which  a  leukocyte  count  is  made  should  always  be 
recorded.  Digestive  leukocytosis  is  most  marked  three 
to  five  hours  after  a  hearty  meal  rich  in  protein.  It  is 
absent  in  pregnancy  and  when  leukocytosis  from  any 
other  cause  exists.  It  is  usually  absent  in  cancer  of  the 
stomach,  a  fact  which  may  be  of  some  help  in  the  diag- 
nosis of  this  condition,  but  repeated  examinations  are 
essential. 

(2)  Pathologic  Polymorphonuclear  Leukocytosis. — 
In  general,  the  response  of  the  leukocytes  to  chemotaxis 


2o6  THE    BLOOD 

is  a  conservative  process.  It  has  been  compared  to  the 
gathering  of  soldiers  to  destroy  an  invader.  This  is 
accomplished  partly  by  means  of  phagocytosis — actual 
ingestion  of  the  enemy — and  partly  by  means  of  chemic 
substances  which  the  leukocytes  produce. 

In  those  diseases  in  which  leukocytosis  is  the  rule  the 
degree  of  leukocytosis  depends  upon  two  factors:  the 
severity  of  the  infection  and  the  resistance  of  the  individual. 
A  well-marked  leukocytosis  usually  indicates  good  resist- 
ance. A  mild  degree  means  that  the  body  is  not  react- 
ing well,  or  else  that  the  infection  is  too  slight  to  call 
forth  much  resistance.  Leukocytosis  may  be  absent 
altogether  when  the  infection  is  extremely  mild,  or  when 
it  is  so  severe  as  to  overwhelm  the  organism  before  it  can 
react.  When  leukocytosis  is  marked,  a  sudden  fall  in 
the  count  may  be  the  first  warning  of  a  fatal  issue. 
These  facts  are  especially  true  of  pneumonia,  diphtheria, 
and  abdominal  inflammations,  in  which  conditions  the 
degree  of  leukocytosis  is  of  considerable  prognostic  value. 

The  classification  here  given  follows  Cabot: 

{a)  Infections  and  Inflammatory. — The  majority  of 
infectious  diseases  produce  leukocytosis.  The  most  not- 
able exceptions  are  influenza,  malaria,  measles,  tuber- 
culosis, except  when  invading  the  serous  cavities  or 
when  complicated  by  mixed  infection,  and  typhoid  fever, 
in  which  leukocytosis  indicates  an  inflammatory  com- 
plication. 

All  inflammatory  and  suppurative  diseases  cause  leu- 
kocytosis, except  when  slight  or  well  walled  off'.  Appen- 
dicitis has  been  studied  with  especial  care  in  this  connec- 
tion, and  the  conclusions  now  generally  accepted  prob- 
ably hold  good  for  most  acute  intra-abdominal  inflam- 


ENUMERATION  OF   LEUKOCYTES  207 

mations.  A  marked  leukocytosis  (20,000  or  more) 
nearly  always  indicates  abscess,  peritonitis,  or  gan- 
grene, even  though  the  clinical  signs  be  slight.  Absence 
of  or  mild  leukocytosis  indicates  a  mild  process,  or  else 
an  overwhelmingly  severe  one ;  and  operation  may  safely 
be  postponed  unless  the  abdominal  signs  are  very  marked. 
On  the  other  hand,  no  matter  how  low  the  count,  an  in- 
creasing leukocytosis — counts  being  made  hourly — indi- 
cates a  spreading  process  and  demands  operation,  regard- 
less of  other  symptoms. 

Leukocyte  counts  alone  are  often  disappointing,  but  are 
of  much  more  value  when  considered  in  connection  with 
a  diferential  count  of  polymorphonuclears.    (See  p.  236.) 

{b)  Malignant  Disease. — Leukocytosis  occurs  in  about 
one-half  of  the  cases  of  malignant  disease.  In  many 
instances  it  is  probably  independent  of  any  secondary 
infection,  since  it  occurs  in  both  ulcerative  and  non- 
ulcerative cases.  It  seems  to  be  more  common  in 
sarcoma  than  in  carcinoma.  Very  large  counts  are  rarely 
noted. 

{c)  Posthemorrhagic. — Moderate  leukocytosis  follows 
hemorrhage  and  disappears  in  a  few  days. 

{d)  Toxic. — This  is  a  rather  obscure  class,  which  in- 
cludes gout,  chronic  nephritis,  acute  yellow  atrophy 
of  the  liver,  ptomain-poisoning,  prolonged  chloroform 
narcosis,  and  quinin-poisoning.  Leukocytosis  may  or 
may  not  occur  in  these  conditions,  and  is  not  important. 

(e)  Drugs. — This  also  is  an  unimportant  class.  Most 
tonics  and  stomachics  and  many  other  drugs  produce  a 
slight  leukoc3^tosis. 

2.  Lymphocyte  Leukocytosis.— This  is  characterized 
by  an  increase  in  the  total  leukocyte  count,  accom- 


2o8  THE   BLOOD 

panied  by  an  increase  in  the  percentage  of  lymphocytes. 
The  word  "  lymphocytosis  "  is  often  used  in  the  same 
sense.  It  is  better,  however,  to  use  the  latter  as  refer- 
ring to  any  increase  in  the  absolute  number  of  lympho- 
cytes, without  regard  to  the  total  count,  since  an  ab- 
solute increase  in  number  of  lymphocytes  is  frequently 
accompanied  by  a  normal  or  subnormal  leukocyte  count, 
owing  to  loss  of  polymorphonuclears. 

Non-phagocytic  leukocytosis  is  probably  due  more  to 
stimulation  of  blood-making  organs  than  to  chemotaxis. 
It  is  less  common,  and  is  rarely  so  marked  as  a  poly- 
morphonuclear leukocytosis.  When  marked,  the  blood 
cannot  be  distinguished  from  that  of  lymphatic  leukemia. 

A  marked  lymphocyte  leukocytosis  occurs  in  pertussis, 
and  is  of  value  in  diagnosis.  It  appears  early  in  the 
catarrhal  stage,  and  persists  until  after  convalescence. 
The  average  leukocyte  count  is  about  17,000,  lympho- 
cytes predominating.  There  is  moderate  lymphocyte 
leukocytosis  in  other  diseases  of  childhood,  as  rickets, 
scurvy,  and  especially  hereditary  syphilis,  where  the 
blood-picture  may  approach  that  of  pertussis.  It 
must  be  borne  in  mind  in  this  connection  that  lympho- 
cytes are  normally  more  abundant  in  the  blood  of  children 
than  in  that  of  adults. 

Slight  lymphocyte  leukocytosis  occurs  in  many  other 
pathologic  conditions,  but  is  of  little  significance. 

B.     Leukemia 
This  is  an  idiopathic  disease  of   the  blood-making 
organs,  which  is  accompanied  by  an  enormous  increase 
in  number  of  leukocytes.     The  leukocyte  count  some- 
times reaches  1,000,000  per  cubic  millimeter,  and  leu- 


ENUMERATION   OF   LEUKOCYTES  209 

kemia  is  always  to  be  suspected  when  it  exceeds  50,000. 
Lower  counts  do  not,  however,  exclude  it.  The  subject 
is  more  fully  discussed  later  (p.  280). 

Method  of  Counting  Leukocytes 
The  leukocytes  are  counted  with  the  Thoma-Zeiss 
instrument,  already  described.  Recently,  several  new 
rulings  of  the  disc  have  been  introduced,  notably  the 
Zappert  and  the  Tiirck  (Fig.  79),  which  give  a  ruled 
area  of  nine  square  millimeters.  They  were  devised  for 
counting  the  leukocytes  in  the  same  specimen  with  the 
red  corpuscles.  The  red  ceUs  are  counted  in  the  usual 
manner,  after  which  all  the  leukocytes  in  the  whole  area 
of  nine  square  millimeters  are  counted;  and  the  number 
in  a  cubic  millimeter  of  undiluted  blood  is  then  easily 
calculated.  Leukocytes  are  easily  distinguished  from 
red  cells,  especially  when  Toisson's  diluting  fluid  is  used. 
This  method  may  be  used  with  the  ordinary  ruling  by 
adjusting  the  microscopic  field  to  a  definite  size,  and 
counting  a  sufficient  number  of  fields,  as  described  later. 
Although  less  convenient,  it  is  more  accurate  to  count  the 
leukocytes  separately,  with  less  dilution  of  the  blood,  as 
follows : 

Technic. — A  larger  drop  of  blood  is  required  than  for 
counting  the  erythrocytes,  and  more  care  in  filling  the  pipet. 
Boggs  has  suggested  a  device  (Fig.  78)  which  enables  one  to 
draw  in  the  blood  more  slowly  and  hence  more  accurately. 
He  cuts  the  rubber  tube  and  inserts  a  Wright  "  throttle." 
This  consists  of  a  section  of  glass  tubing  in  which  a  capillary 
tube  drawn  out  to  a  fine  thread  is  cemented  with  sealing  wax. 
After  sealing  in  place  the  tip  is  broken  off  with  forceps,  so 
that  upon  gentle  suction  it  will  just  allow  air  to  pass. 

14 


2IO 


THE   BLOOD 


Use  the  pipet  with  1 1  engraved  above  the  bulb.  Suck  the 
blood  to  the  mark  0.5  or  i.o,  and  the  diluting  fluid  to  the 
mark  11.  This  gives  a  dilution  of  i :  20  or  1:10,  respectively. 
The  dilution  of  i  :  20  is  easier  to  make.  Mix  well  by  shaking 
in  all  directions  except  in  the  long  axis  of  the  pipet;  blow  out 
two  or  three  drops,  place  a  drop  in  the  counting  chamber, 
and  adjust  the  cover  as  already  described  (p.  196). 


Fig.  78. — Boggs'  "  throttle  control "  for  blood-counting  pip>et,  and  enlarged  diagram  show- 
ing construction  of  the  throttle. 


Examine  with  a  low  power  to  see  that  the  cells  are  evenly 
distributed.  Count  with  the  16  mm.  objective  and  a  high 
eye-piece,  or  with  the  long-focus  4  mm.  and  a  low  eye-piece. 
An  8  mm.  objective  will  be  found  very  satisfactory  for  this 
purpose.  As  one  gains  experience  one  will  rely  more  upon 
the  lower  powers. 

With  the  ordinary  ruling  of  the  disc,  count  all  the  leuko- 
cytes in  the  large  square,  multiply  by  10  to  find  the  number  in 


ENUMERATION  OF   LEUKOCYTES 


211 


I  c.mm.  of  diluted  blood,  and  by  the  dilution  to  find  the 
number  per  c.mm.  of  undiluted  blood.  In  every  case  at  least 
200  leukocytes  must  be  counted  as  a  basis  for  calculation,  and 
it  is  much  better  to  coimt  500.  This  will  necessitate  exam- 
ination of  several  drops  from  the  pipet.     With  the  Zappert 


Fig.  79. — Turck  nxling  of  counting  chamber. 


and  Tiirck  rulings  a  sufficient  number  can  usually  be  counted 
in  one  drop,  but  the  opportunity  for  error  is  very  much  greater 
when  only  one  drop  is  examined. 

In  routine  work  the  author's  modification  of  the  "circle  " 
method  is  very  satisfactory.    It  requires  a  4  mm.  objective, 


212 


THE    BLOOD 


and  is,  therefore,  especially  desirable  for  beginners,  who  are 
usually  unable  accurately  to  identify  leukocytes  with  a  lower 
power.  The  student  is  frequently  confused  by  particles  of 
dirt,  remains  of  red  cells,  and  yeast  cells  which  sometimes 
grow  in  the  diluting  fluid.     Draw  out  the  sliding  tube  of  the 


1           1 

1               > 

--r r 

.     4.             1 

,___ 

r  - 

1 

r 

1       ■       1              r 
ill              1 

r r-  ""--T r-- 

L      1       1             1 

1           1 

--T -t- 

1            1 

1 

1 t-  - 

1 

1 

r  -+ 

1 

r  — 

1         1     !     '         ' 
r---T---T--,--;---;-- 

1                 r        ,        1 

1 
-    -  -t  - 

r -t-  - 

hs 

i  .  1      ; 

I.             -              4^             1-             L                             I 

\ 

s^              1 

\        1 

/ 

\  1 

■  / 

1                        /    L 

\  \     ; 

1 

1                             \ 

( 

'y.iV-'X:. 

1                             1    \ 
-            1                            1             \ 

-:     1 1-- 

1                             1                    \ 
1                             1 

--4- ^---1 

1            1 

1            1 
__4- 1--..1 

1            1 

1            1 

1            1 

Fig.  8o. — Size  o 

^, 

y 

V 

field  re 

-  -1 
■  - 
quii 

ed  i 

, 1 

. ^ 

n  count 

\- ■ 

ng  leuk 

ocytes  a 

^---l-r-^---|- 
1    :    .        ! 

s  described  in  the  text. 

microscope  until  the  field  of  vision  is  such  as  shown  in  Fig. 
8o.  One  side  of  the  field  is  tangent  to  one  of  the  ruled  lines, 
A,  while  the  opposite  side  just  cuts  the  comers,  B  and  C,  of 
the  seventh  squares  in  the  rows  above  and  below  the  diameter 
line.     When  once  adjusted,  a  scratch  is  made  upon  the  draw- 


ENUMERATION   OF   BLOOD-PLAQUES  213 

tube,  SO  that  for  future  counts  the  tube  has  only  to  be  drawn 
out  to  the  mark.  The  area  of  this  microscopic  field  is  one- 
tenth  of  a  square  millimeter.  With  a  dilution  of  i  :  20,  count 
the  leukocytes  in  20  such  fields  upon  different  parts  of  the  disc 
without  regard  to  the  ruled  lines,  and  to  their  sum  add  two 
ciphers.  With  dilution  of  i  :  10,  count  10  such  fields,  and 
add  two  ciphers.  Thus,  with  i  :  10  dilution,  if  150  leukocytes 
were  counted  in  10  fields,  the  leukocyte  count  would  be  15,000 
per  c.mm.  To  compensate  for  possible  unevenness  of  dis- 
tribution, it  is  best  to  count  a  row  of  fields  horizontally  and  a 
row  vertically  across  the  disc.  This  method  is  applicable 
to  any  degree  of  dilution  of  the  blood,  and  is  simple  to  re- 
member :  one  always  counts  a  number  of  fields  equal  to  the 
number  of  times  the  blood  has  been  diluted,  and  adds  two  ciphers. 

It  is  sometimes  impossible  to  obtain  the  proper  size  of 
field  with  the  objectives  and  eye-pieces  at  hand.  In  such  case 
place  a  cardboard  disc  with  a  circular  opening  upon  the  dia- 
phragm of  the  eye-piece,  and  adjust  the  size  of  the  field  by 
drawing  out  the  tube.  The  circular  opening  can  be  cut  with 
a  cork-borer. 

Diluting  Fluids. — The  diluting  fluid  should  dissolve  the 
red  corpuscles  so  that  they  will  not  obscure  the  leukocytes. 
The  simplest  fluid  is  a  0.5  per  cent,  solution  of  acetic  acid. 
More  satisfactory  is  the  following:  glacial  acetic  acid,  i  c.c; 
I  per  cent,  aqueous  solution  of  gentian-violet,  i  c.c. ;  distilled 
water,  100  c.c.     These  solutions  must  be  filtered  frequently. 

VI.  ENUMERATION  OF  BLOOD-PLAQUES 

The  av^erage  normal  number  of  plaques  is  variously 
given  as  200,000  to  700,000  per  cubic  millimeter  of 
blood.  Many  of  the  counts  were  obtained  by  workers 
who  used  unreliable  methods.  Using  their  new  method, 
Wright  and  Kinnicutt  find  the  normal  average  to  range 
from   263,000  to  336,000.     Physiologic  variations  are 


214  THE   BLOOD 

marked;  thus,  the  number  increases  as  one  ascends  to  a 
higher  altitude,  and  is  higher  in  winter  than  in  summer. 
There  are  unexplained  variations  from  day  to  day; 
hence  a  single  abnormal  count  should  not  be  taken  to 
indicate  a  pathologic  condition. 

Pathologic  variations  are  often  very  great.  Owing  to 
lack  of  knowledge  as  to  the  origin  of  the  platelets  and  to 
the  earlier  imperfect  methods  of  counting,  the  clinical 
significance  of  these  variations  is  uncertain.  The  fol- 
lowing facts  seem,  however,  to  be  established: 

(a)  In  acute  infectious  diseases  the  number  is  sub- 
normal or  normal.  If  the  fever  ends  by  crisis,  the  crisis 
is  accompanied  by  a  rapid  and  striking  increase. 

(b)  In  secondary  anemia  plaques  are  generally  in- 
creased, although  sometimes  decreased  In  pernicious 
anemia  they  are  always  greatly  diminished,  and  an 
increase  should  exclude  the  diagnosis  of  this  disease. 

(c)  They  are  decreased  in  chronic  lymphatic  leukemia, 
and  greatly  increased  in  the  myelogenous  form. 

(d)  In  purpura  haemorrhagica  the  number  is  enor- 
mously diminished. 

Blood-plaques  are  difficult  to  count,  owing  to  the 
rapidity  with  which  they  disintegrate  and  to  their  great 
tendency  to  adhere  to  any  foreign  body  and  to  each 
other. 

Method  of  Kemp,  Calhoun,  and  Harris. — Wash  the 
finger  well  and  allow  a  few  minutes  to  elapse  for  the  circu- 
lation to  become  normal.  Prick  the  finger  lightly  with  a 
blood-lancet,  regulating  the  depth  of  the  puncture  so  that 
the  blood  will  not  flow  without  gentle  pressure.  Quickly 
dip  a  clean  glass  rod  into  a  vessel  containing  diluting  and 
fixing  fluid,  and  place  two  or  three  good-sized  drops  upon  the 


ENUMERATION   OF  BLOOD-PLAQUES  215 

finger  over  the  puncture.  Then  exert  gentle  pressure  above 
the  puncture  so  that  a  small  drop  of  blood  will  exude  into  the 
fluid.  Mix  the  two  by  passing  the  rod  lightly  several  times 
over  the  surface  of  the  blended  drop.  (Some  workers  first 
place  a  drop  of  the  fluid  upon  the  finger  and  then  make  the 
puncture  through  it,  this  necessitating  less  care  as  to  depth  of 
the  puncture.)  Now  transfer  a  drop  of  the  diluted  blood  from 
the  finger  to  a  watch-glass  which  contains  two  or  three  drops 
of  the  fluid,  and  mix  well.  From  this,  transfer  a  drop  to  the 
counting  slide  of  the  Thoma-Zeiss  hemocytometer,  and  cover. 
An  ordinary  thin  cover  will  answer  for  this  purpose,  and  is 
preferable  because  it  allows  the  use  of  a  higher  power  object- 
ive. Allow  the  slide  to  stand  for  at  least  five  minutes,  and 
then  with  a  4  mm.  or  higher  objective  count  the  plaques 
and  the  red  corpuscles  in  a  definite  number  of  squares.  At 
least  100  plaques  must  be  counted.  The  number  of  red  cor- 
puscles per  cubic  millimeter  of  blood  having  been  previously 
ascertained  in  the  usual  manner  (p.  195),  the  number  of 
plaques  can  easily  be  calculated  by  the  following  equation: 

r  -.p  -.-.R-.P  ;  andP  =  ^Jl_?. 

r  represents  the  number  of  red  corpuscles  in  any  given 
number  of  squares;  p,  the  number  of  plaques  in  the  same 
squares;  R,  the  total  number  of  red  corpuscles  per  c.mm.  of 
blood;  and  P,  the  number  of  plaques  per  c.mm. 

Beginners  are  apt  to  take  too  much  blood  and  not  to  dilute 
it  enough.  Best  results  are  attained  when  there  are  only  one 
or  two  plaques  in  a  small  square.  With  insufladent  dilution, 
the  platelets  are  more  or  less  obscured  by  the  red  cells. 

The  following  diluting  and  fixing  fluid  is  recommended: 

Formalin 10  c.c. 

I  per  cent,  aqueous  solution  sodium  chlorid 150  c.c. 

(Color  with  methyl-violet  if  desired.) 


2l6  THE    BLOOD 

This  fluid  is  cheap  and  easily  prepared,  and  keeps  indefi- 
nitely. It  fixes  the  plaques  quickly  without  clumping,  and 
does  not  clump  nor  decolorize  the  reds.  It  has  a  low  specific 
gravity,  and  hence  allows  the  j^laques  to  settle  upon  the  ruled 
area  along  with  the  reds.  Fluids  of  high  specific  gravity 
cause  the  plaques  to  float  so  that  they  do  not  appear  in  the 
same  plane  with  the  reds  and  the  ruled  lines. 

Method  of  Wright  and  Kinnicutt. — This  new  method  is 
simple,  appears  to  be  accurate,  and  certainly  yields  uniform 
results. 

The  plaques  are  counted  with  the  Thoma-Zeiss  hemocy- 
tometer  already  described,  using  a  dilution  of  i  :  loo.  The 
diluting  fluid  consists  of  two  parts  of  an  aqueous  solution  of 
"  brilliant  cresyl  blue  "  (i  :  300)  and  three  parts  of  an  aqueous 
solution  of  potassium  cyanid  (i  :  1400).  These  two  solutions 
must  be  kept  in  separate  bottles  and  mixed  and  filtered  im- 
mediately before  using.  After  the  blood  is  placed  in  the 
counting-chamber  it  is  allowed  to  stand  for  ten  minutes  or 
longer  in  order  that  the  plaques  may  settle.  The  plaques 
appear  as  rounded,  lilac-colored  bodies;  the  reds  are  decolor- 
ized, appearing  only  as  shadows. 

The  leukocytes  are  stained  and  may  be  counted  at  the 
same  time. 

VII.  STUDY  OF  STAINED  BLOOD 

A.     Making  and  Staining  Blood-films 

1 .  Spreading  the  Film.— Thin,  even  films  are  essential 
to  accurate  and  pleasant  work.  They  more  than  com- 
pensate for  the  time  spent  in  learning  to  make  them. 
There  are  certain  requisites  for  success  with  any  method : 
(a)  The  slides  and  covers  must  be  perfectly  clean: 
thorough  washing  with  soap  and  water  and  rubbing 
with  alcohol  will  usually  suffice;  (b)  the  drop  of  blood 


STUDY   OF  STAINED  BLOOD  217 

must  not  be  too  large ;  (c)  the  work  must  be  done  quickly, 
before  coagulation  begins. 

The  blood  is  obtained  from  the  finger-tip  or  the  lobe 
of  the  ear,  as  for  a  blood  count;  only  a  very  small  drop  is 
required. 

Ehrlich's  Two  Cover-glass  Method. — This  method  is  very 
widely  used,  but  considerable  practice  is  required  to  get  good 
results.  Touch  a  cover-glass  to  the  top  of  a  small  drop  of 
blood,  and  place  it,  blood  side  down,  upon  another  cover- 


Fig.  81. — Spreading  the  film:  two  cover-glass  method. 

glass.  If  the  drop  be  not  too  large,  and  the  covers  be  per- 
fectly clean,  the  blood  will  spread  out  in  a  very  thin  layer. 
Just  as  it  stops  spreading,  before  it  begins  to  coagulate, 
pull  the  covers  quickly  but  firmly  apart  on  a  line  parallel  to 
their  plane  (Fig.  81).  It  is  best  to  handle  the  covers  with 
forceps,  since  the  moisture  of  the  fingers  may  produce  arti- 
facts. 

Two-slide  Method. — Take  a  small  drop  of  blood  upon  a 
clean  slide  about  5  inch  from  the  end.  Place  the  end  of  a 
second  slide  against  the  surface  of  the  first  at  an  angle  of 
45°,  and  push  it  up  against  the  drop  of  blood,  which  will 
immediately  run  across  the  end,  filling  the  angle  between  the 


2l8 


THE   BLOOD 


two  slides.  Now  draw  the  "  spreader  slide  "  back  along  the 
other.  The  blood  will  follow.  The  thickness  of  the  smear 
can  be  regulated  by  changing  the  angle. 


Fig.  82. — Spreading  the  film:  two-slide  method. 

Cigarette-paper  Method. — This  gives  better  results  in  the 
hands  of  the  inexperienced  than  any  of  the  methods  in  general 


Fig.  83. — Spreading  the  film.     Cigarette-paper  method  applied  to  cover-glasses. 

use,  and  may  be  used  with  either  slides  or  covers.   A  very  thin 
paper,  such  as  the  "  Zig-zag "  brand,  is  best.    Ordinary 


STUDY   OF   STAINED   BLOOD  219 

cigarette  paper  and  thin  tissue-paper  will  answer,  but  do  not 
give  nearly  so  good  results. 

Cut  the  paper  into  strips  about  f  inch  wide,  across  the  ribs. 
Pick  up  one  of  the  strips  by  the  gummed  edge,  and  touch  its 
opposite  end  to  the  drop  of  blood.  Quickly  place  the  end 
which  has  the  blood  against  a  slide  or  a  large  cover-glass  held 
in  a  forceps.  The  blood  will  spread  along  the  edge  of  the 
paper.  Now  draw  the  paper  evenly  across  the  slide  or  cover. 
A  thin  film  of  blood  will  be  left  behind  (Fig.  83). 

The  films  may  be  allowed  to  dry  in  the  air,  or  may  be 
dried  by  gently  heating  high  above  a  flame  (where  one 
can  comfortably  hold  the  hand).  Such  films  will  keep 
for  years,  but  for  some  stains  they  must  not  be  more 
than  a  few  weeks  old.  They  must  be  kept  away  from 
flies — a  fly  can  work  havoc  with  a  film  in  a  few  minutes. 

2.  Fixing  the  Film.— In  general,  films  must  be  "fixed" 
before  they  are  stained.  Fixation  may  be  accomplished 
by  chemicals  or  by  heat.  Those  stains  which  are  dis- 
solved in  methyl-alcohol  combine  fixation  with  the  staining 
process. 

Chemic  Fixation. — Soak  the  film  five  to  fifteen  minutes 
in  pure  methyl-alcohol,  or  one-half  hour  or  longer  in  equal 
parts  of  absolute  alcohol  and  ether.  One  minute  in  i  per  cent, 
formalin  in  alcohol  is  preferred  by  some.  Chemic  fixation  may 
precede  eosin-methylene-blue  and  other  simple  stains. 

Heat  Fixation. — This  may  precede  any  of  the  methods 
which  do  not  combine  fixation  with  the  staining  process;  it 
must  be  used  with  Ehrlich's  triple  stain.  The  best  method  is 
to  place  the  film  in  an  oven,  raise  the  temperature  to  150°  C, 
and  allow  to  cool  slowly.  Without  an  oven,  the  proper 
degree  of  fixation  is  difficult  to  attain.  Kowarsky  has  de- 
vised a  small  plate  of  two  layers  of  copper  (Fig.  84),  upon 


220  THE   BLOOD 

which  the  films  are  placed  together  with  a  crystal  of  urea. 
The  plate  is  heated  over  a  flame  until  the  urea  melts,  and  is 
then  set  aside  to  cool.  This  is  said  to  give  the  proper  degree 
of  fixation,  but  in  the  writer's  experience  the  films  have  always 
been  underheated.  He  obtains  better  results  by  use  of  tar- 
taric acid  crystals  (melting-point,  i68°-i7o°  C).  The  pla.te, 
upon  which  have  been  placed  the  cover-glasses,  film  side 
down,  and  a  crystal  of  the  acid,  is  heated  over  a  low  flame  until 
the  crystal  has  completely  melted.  It  should  be  held  suffi- 
ciently high  above  the  flame  that  the  heating  will  require  five 
to  se\en  minutes.  The  covers  are  then  removed.  Freshly 
made  films  of  normal  blood  should  be  allowed  to  remain  upon 


Fig.  84. — Kowarsky's  plate  for  fixing  blood  (Klopstock  and  Kowarsky). 

the  plate  for  a  minute  or  two  after  heating  has  ceased. 
Fresh  films  require  more  heat  than  old  ones,  and  normal  blood 
more  than  the  blood  of  pernicious  anemia  and  leukemia. 

Fixation  by  passing  the  film  quickly  through  a  flame  about 
twenty  times,  as  is  often  done  in  routine  work,  is  not  recom- 
mended for  beginners. 

3.  Staining  the  Film. — The  anilin  dyes,  which  are 
extensively  used  in  blood  work,  are  of  two  general  classes: 
basic  dyes,  of  which  methylene-blue  is  the  t^pe;  and  acid 
dyes,  of  which  eosin  is  the  type.  Nuclei  and  certain 
other  structures  in  the  blood  are  stained  by  the  basic 
dyes,  and  are  hence  called  basophilic.     Certain  struc- 


STUDY   OF   STAINED   BLOOD  221 

tures  take  up  only  add  dyes,  and  are  called  acidophilic, 
oxyphilic,  or  eosinophilic.  Certain  other  structures  are 
stained  only  by  combinations  of  the  two,  and  are  called 
neutrophilic.  Recognition  of  these  staining  properties 
marked  the  beginning  of  modern  hematology. 

(i)  Eosin  and  Methylene-blue. — In  many  instances 
this  stain  will  give  all  the  information  desired.  It  is 
especially  useful  in  studying  the  red  corpuscles.  Nuclei, 
basophilic  granules,  and  all  blood  parasites  are  blue; 
erythrocytes  are  red  or  pink;  eosinophilic  granules,  bright 
red.  Neutrophihc  granules  and  blood-plaques  are  not 
stained. 

(i)  Fix  the  film  by  heat  or  chemicals. 

(2)  Stain  about  five  minutes  with  0.5  per  cent,  alcoholic 
solution  of  eosin,  diluted  one-half  with  water. 

(3)  Rinse  in  water,  and  dry  between  filter-papers. 

(4)  Stain  one-half  to  one  minute  with  saturated  aqueous 
solution  of  methylene-blue. 

(5)  Rinse  well,  dry,  and  mount.  Films  upon  slides  may 
be  examined  with  an  oil-immersion  objective  without  a  cover- 
glass. 

(2)  Ehrlich's  Triple  Stain. — This  has  been  the  stand- 
ard blood-stain  for  many  years,  but  is  now  little  used. 
It  is  probably  the  best  for  neutrophihc  granules.  It  is 
difficult  to  make,  and  should  be  purchased  ready  pre- 
pared from  a  reHable  dealer.  Nuclei  are  stained  blue 
or  greenish  blue;  erythrocytes,  orange;  neutrophilic 
granules,  violet;  and  eosinophilic  granules,  copper  red. 
Basophilic  granules  and  blood-plaques  are  not  stained. 

Success  in  staining  depends  largely  upon  proper  fixa- 
tion.    The  film  must  be  carefully  fixed  by  heat:   under- 


222  THE   BLOOD 

heating  causes  the  erythrocytes  to  stain  red;  overheat- 
ing, pale  yellow.  The  staining  fluid  is  applied  for  five 
to  fifteen  minutes,  and  the  preparation  is  rinsed  quickly 
in  water,  dried,  and  mounted.  Subsequent  application 
of  Lofiler's  methylene-blue  for  one-half  to  one  second  will 
bring  out  the  basophilic  granules  and  improve  the 
nuclear  staining,  but  there  is  considerable  danger  of 
overstaining. 

(3)  Polychrome  Methylene-blue  Eosin  Stains. — These 
stains,  outgrowths  of  the  original  Romanowsky  method, 
have  largely  displaced  other  blood-stains  for  clinical 
purposes.  They  stain  differentially  every  normal  and 
abnormal  structure  in  the  blood.  Most  of  them  are 
dissolved  in  methyl  alcohol  and  combine  the  fbdng  with 
the  staining  process.  Numerous  methods  of  preparing 
and  applying  these  stains  have  been  devised.  Two 
only  need  be  given  here:  Wright's  stain  and  Harlow's 
stain : 

Wright's  Stain. — This  is  one  of  the  best  and  is  the 
most  widely  used  in  this  country.  The  practitioner 
will  find  it  best  to  purchase  the  stain  ready  prepared. 
Most  microscopic  supply-houses  carry  it  in  stock. 
Wright's  most  recent  directions  for  its  preparation  and 
use  are  as  follows:' 

Preparation. — To  a  0.5  per  cent,  aqueous  solution  of 
sodium  bicarbonate  add  methylene-blue  (B.  X.  or  "  medicin- 
ally pure  ")  in  the  proportion  of  i  gm.  of  the  dye  to  each  100 
c.c.  of  the  solution.  Heat  the  mixture  in  a  steam  sterilizer 
at  100°  C.  for  one  full  hour,  counting  the  time  after  the  ster- 
ilizer has  become  thoroughly  heated.  The  mixture  is  to  be 
contained  in  a  flask,  or  flasks,  of  such  size  and  shape  that  it 
^Journal  of  the  American  Medical  Association,  Dec.  3,  1910. 


STUDY  OF   STAINED   BLOOD  223 

forms  a  layer  not  more  than  6  cm.  deep.  After  heating, 
allow  the  mixture  to  cool,  placing  the  flask  in  cold  water, 
if  desired,  and  then  filter  it  to  remove  the  precipitate  which 
has  formed  in  it.  It  should,  when  cold,  have  a  deep  purple- 
red  color  when  viewed  in  a  thin  layer  by  transmitted  yellowish 
artificial  light.    It  does  not  show  this  color  while  it  is  warm. 

To  each  100  c.c.  of  the  filtered  mixture  add  500  c.c.  of  a 
0.1  per  cent,  aqueous  solution  of  "  yellowish  water-soluble  " 
eosin  and  mix  thoroughly.  Collect  the  abundant  precipitate 
which  immediately  appears  on  a  filter.  When  the  precipitate 
is  dry,  dissolve  it  in  methylic  alcohol  (Merck's  "  reagent ") 
in  the  proportion  of  o.i  gm.  to  60  c.c.  of  the  alcohol.  In 
order  to  facilitate  solution,  the  precipitate  is  to  be  rubbed  up 
with  the  alcohol  in  a  porcelain  dish  or  mortar  with  a  spatula 
or  pestle.  This  alcoholic  solution  of  the  precipitate  is  the 
staining  fluid. 

Application. — i.  Cover  the  film  with  a  noted  quantity  of 
the  staining  fluid  by  means  of  a  medicine-dropper. 

2.  After  one  minute  add  to  the  staining  fluid  on  the  film 
the  same  quantity  of  distilled  water  by  means  of  a  medicine- 
dropper  and  allow  the  mixture  to  remain  for  two  or  three 
minutes,  according  to  the  intensity  of  the  staining  desired. 
A  longer  period  of  staining  may  produce  a  precipitate. 
Eosinophilic  granules  are  best  brought  out  by  a  short  period 
of  staining. 

The  quantity  of  the  diluted  fluid  on  the  preparation  should 
not  be  so  large  that  some  of  it  runs  off. 

3.  Wash  the  preparation  in  water  for  thirty  seconds  or 
until  the  thinner  portions  of  the  film  become  yellow  or  pink 
in  color. 

4.  Dry  and  mount  in  balsam. 

The  stain  is  more  conveniently  applied  upon  cover- 
glasses  than  upon  slides.  Films  much  more  than  a  month 
old  do  not  stain  well.     In  some  localities  ordinary  tap- 


224  iHE    BLOOD 

water  will  answer  both  for  diluting  the  stain  and  for 
washing  the  film;  in  others,  distilled  water  must  be  used. 
Different  lots  of  Wright's  fluid  vary,  and  a  few  prelimin- 
ary stains  should  be  made  with  each  lot  to  learn  its 
peculiarities. 

When  properly  applied,  Wright's  stain  gives  the  fol- 
lowing picture  (Plate  VI):  erythrocytes,  yellow  or  pink; 
nuclei,  various  shades  of  bluish  purple;  neutrophilic 
granules,  reddish  Hlac;  eosinophilic  granules,  bright  red; 
basophilic  granules  of  leukocytes  and  degenerated  red 
corpuscles,  very  dark  bluish  purple;  blood-plaques, 
dark  lilac;  bacteria,  blue.  The  cytoplasm  of  lymphocytes 
is  generally  robin's-egg  blue;  that  of  the  large  mononu- 
clears may  have  a  faint  bluish  tinge.  Malarial  parasites 
stain  characteristically:  the  cytoplasm,  sky-blue;  the 
chromatin,  reddish  purple. 

Harlow's  Stain. — Probably  the  simplest  modification  of 
the  Romanowsky  stain,  both  in  preparation  and  method 
of  use,  is  that  devised  by  W.  P.  Harlow  of  the  University 
of  Colorado.  It  differentiates  granules  particularly 
well,  but  is  not  so  satisfactory  for  demonstrating  slight 
grades  of  polychromatophilia,  because  it  usually  gives  all 
the  red  cells  a  faint  bluish  tinge. 

Preparation. — The  stain  consists  of  two  solutions  used 
separately: 

No.  I.  Eosin,  yellowish,  water  soluble  (Griibler) .      i  gram 

Methyl  alcohol  (Merck's  reagent) loo  c.c. 

No.  2.   Methylene-blue    ("  B.   X."   or   Ehrlich's 

rectified)  (Griibler) i  gram 

Methyl  alcohol  (Merck's  reagent) loo  c.c. 

Application. — (i)  Stain  the  film  without  previous  fixation 
for  one  minute  with  the  eosin  solution. 


STUDY  OF  STAINED  BLOOD  22$ 

(2)  Shake  off  the  excess,  allowing  a  very  little  to  remain, 
and  at  once  cover  with  the  methylene-blue  solution  for  one 
or  two  minutes. 

(3)  Rinse  quickly  in  distilled  water,  dry,  and  mount. 

It  is  well  known  that  pathologic  bloods  will  sometimes 
not  stain  well  with  fluids  which  are  satisfactory  for 
normal  bloods.  Doctors  Peebles  and  Harlow  have  shown 
that  the  various  polychrome  methylene-blue-eosin  stains 
can  be  modified  to  suit  any  blood  by  adding  a  little 
alkali  or  acid.  The  alkali  used  is  a  weak  solution  of 
"  potassium  hydrate  by  alcohol  "  in  methyl  alcohol;  the 
acid,  glacial  acetic  in  methyl  alcohol.  In  the  case  of  the 
Harlow  stain  it  is  added  to  the  methylene-blue  solution 
only.  The  alkali  solution  also  serves  to  "  correct  "  old 
fluids  which,  by  reason  of  development  of  formic  acid 
in  the  methyl  alcohol,  do  not  stain  sufficiently  with  the 
blue.  In  general  a  stain  is  satisfactory  when  both  nuclei 
and  neutrophilic  granules  are  clearly  defined. 

B.     Study  of  Stained  Films 

Much  can  be  learned  from  stained  blood-films.  They 
furnish  the  best  means  of  studying  the  morphology  of  the 
blood  and  blood  parasites,  and,  to  the  experienced,  they 
give  a  fair  idea  of  the  amount  of  hemoglobin  and  the 
number  of  red  and  white  corpuscles.  An  oil-immersion 
objective  is  required. 

1.  Erythrocytes.— Normally,  the  red  corpuscles  are 
acidophilic.  The  colors  which  they  take  with  different 
stains  have  been  described.  When  not  crowded  to- 
gether, they  appear  as  circular,  homogeneous  discs,  of 
nearly  uniform  size,  averaging  7.5  fi  in  diameter  (Fig. 
104).    The  center  of  each  is  somewhat  paler  than  the 

15 


226  THE  BLOOD 

periphery.  The  degree  of  pallor  furnishes  a  rough  index 
to  the  amount  of  hemoglobin  in  the  corpuscle.  As 
hemoglobin  is  diminished,  the  central  pale  area  becomes 
larger  and  paler,  producing  the  so-called  "  pessary 
forms  "  in  which  only  the  periphery  of  the  cell  is  apparent. 
These  forms  indicate  a  low  color  index  and  are  most 
abundant  in  chlorosis.  Red  cells  are  apt  to  be  crenated 
when  the  film  has  dried  too  slowly. 

Pathologically,  red  corpuscles  vary  in  size  and  shape, 
staining  properties,  and  structure. 

(i)  Variations  in  Size  and  Shape  (See  Plate  IX  and 
Fig.  104). — The  cells  may  be  abnormally  small  (called 
microcytes,  5  ;(/  or  less  in  diameter);  abnormally  large 
{macrocytes,  10  to  12  u);  or  extremely  large  (megalocytes, 
12  to  20  ^). 

Variation  in  shape  is  often  very  marked.  Oval,  pyri- 
form,  caudate,  saddle-shaped,  and  club-shaped  corpus- 
cles are  common  (Fig.  85).  They  are  called  poikilocytes, 
and  their  presence  is  spoken  of  as  poikilocytosis. 

Red  corpuscles  which  vary  from  the  normal  in  size  and 
shape  are  present  in  most  symptomatic  anemias,  and  in 
the  severer  grades  are  often  very  numerous.  Irregular- 
ities are  particularly  conspicuous  in  leukemia  and  pernic- 
ious anemia,  where,  in  some  instances,  a  normal  erythro- 
cyte is  the  exception.  In  pernicious  anemia  there  is  a 
decided  tendency  to  large  size  and  oval  forms,  and  mega- 
locytes are  rarely  found  in  any  other  condition. 

(2)  Variations  in  Staining  Properties  (See  Plate 
IX). — These  include  polychromatophilia,  basophiHc 
degeneration,  and  malarial  stippling.  With  exception 
of  polychromatophilia  they  are  probably  degenerative 
changes. 


STUDY  OF  STAINED  BLOOD 


227 


(a)  Polychromatophilia. — Some  of  the  corpuscles  par- 
tially lose  their  normal  affinity  for  acid  stains,  and  take 
the  basic  stain  to  greater  or  less  degree.  Wright's  stain 
gives  such  cells  a  faint  bluish  tinge  when  the  condition  is 
mild,  and  a  rather  deep  blue  when  severe.  Sometimes 
only  part  of  a  cell  is  affected.  A  few  polychromatophilic 
corpuscles  can  be  found  in  marked  symptomatic  anemias. 


Fig.  85. — Abnormal  red  corpuscles:  A,  Poikilocytosis;  B,  basophilic  granular  degenera- 
tion; C,  malarial  stippling,  the  cell  also  containinij  a  tertian  parasite  ( X  1000)  (courtesy  of 
Dr.  W.  P.  Harlow). 


They  occur  most  abundantly  in  malaria,  leukemia,  and 
pernicious  anemia. 

Polychromatophilia  has  been  variously  interpreted. 
It  is  thought  by  many  to  be  evidence  of  youth  in  a  cell, 
and  hence  to  indicate  an  attempt  at  blood  regeneration. 
There  are  probably  several  forms  referable  to  different 
causes. 

{h)  Basophilic  Granular  Degeneration  {Degeneration  oj 
Grawitz). — This  is  characterized  by  the  presence,  within 


228  THE   BLOOD 

the  corpuscle,  of  basophilic  granules  which  vary  in  size 
from  scarcely  visible  points  to  granules  as  large  as  those 
of  basophilic  leukocytes  (Fig.  85).  The  number  present 
in  a  red  cell  commonly  varies  in  inverse  ratio  to  their 
size.  They  stain  deep  blue  with  Wright's  stain;  not  at 
all  with  Ehrlich's  triple  stain.  The  cell  containing  them 
may  stain  normally  in  other  respects,  or  it  may  exhibit 
polychromatophilia. 

Numerous  cells  showing  this  degeneration  are  com- 
monly found  in  chronic  lead-poisoning,  of  which  they 


Fig.  86. — Normoblasts  from  cases  of  secondary  anemia  and  leukemia  (X  looo)  (photo- 
graphs by  the  author). 


were  at  one  time  thought  to  be  pathognomonic.  Except 
in  this  disease,  the  degeneration  indicates  a  serious  blood 
condition.  It  occurs  in  well-marked  cases  of  pernicious 
anemia  and  leukemia,  and,  much  less  commonly,  in  very 
severe  symptomatic  anemias. 

(c)  Malarial  Stippling. — This  term  has  been  applied 
to  the  finely  granular  appearance  often  seen  in  red  cor- 
puscles, which  harbor  malarial  parasites  (Plates  VI  and 
VII  and  Fig.  85).  It  is  commonly  classed  with  the  degen- 
eration just  described,  but  is  probably  distinct.  Not 
all  stains  will  show  it.     With  Wright's  stain  it  can  be 


STUDY  OF  STAINED  BLOOD  229 

brought  out  by  staining  longer  and  washing  less  than 
for  the  ordinary  blood-stain.  The  minute  granules  stain 
reddish  purple. 

(3)  Variations  in  Structure. — The  most  important  is 
the  presence  of  a  nucleus  (Plates  VI  and  IX  and  Fig. 
86).  Nucleated  red  corpuscles,  or  erytkroblasts ,  are 
classed  according  to  their  size:  microblasts,  5  |M  or  less  in 
diameter;  normoblasts,  5  to  10  a;  and  megaloblasts,  above 
10  u.  Microblasts  and  normoblasts  contain  one,  rarely 
two,  small  round,  sharply  defined,  deeply  staining  nuclei, 


Fig.  87. — Megaloblasts  from  a  case  of  pernicious  anemia  ( X    1000)  (courtesy  of  Dr. 
W.  P.  Harlow). 

often  located  eccentrically.  Occasionally  the  nucleus  is 
irregular  in  shape,  "  clover-leaf  "  forms  being  not  infre- 
quent. The  megaloblast  (Fig.  87)  is  probably  a  distinct 
cell,  not  merely  a  larger  size  of  the  normoblast.  Its 
nucleus  is  large,  stains  rather  palely,  has  a  delicate 
chromatin  network,  and  often  shows  evidences  of  degen- 
eration (karyorrhexis,  etc.).  In  ordinary  work,  however, 
it  is  safer  to  base  the  distinction  upon  size  than  upon 
structure.  Any  nucleated  red  cell,  but  especially  the 
megaloblast,  may  exhibit  polychromatophilia. 

Normally,  erythroblasts  are  present  only  in  the  blood 


230  THE   BLOOD 

of  the  fetus  and  of  very  young  infants.  In  the  adult, 
their  presence  in  the  circulating  blood  denotes  an  excess- 
ive demand  upon  the  blood-forming  organs  to  regenerate 
lost  or  destroyed  red  corpuscles.  In  response  to  this 
demand,  immature  and  imperfectly  formed  cells  are 
thrown  into  the  circulation.  Their  number,  therefore, 
is  an  indication  of  the  extent  to  which  the  bone-marrow 
reacts  rather  than  of  the  severity  of  the  disease.  Nor- 
moblasts occur  in  severe  symptomatic  anemia,  leukemia, 
and  pernicious  anemia.  They  are  most  abundant  in 
myelogenous  leukemia.  While  always  present  in  per- 
nicious anemia,  they  are  often  difficult  to  find.  Megalo- 
blasts  are  found  in  pernicious  anemia,  and  with  extreme 
rarity  in  any  other  condition.  They  here  almost  inva- 
riably exceed  the  normoblasts  in  number,  which  is  one  of 
the  distinctive  features  of  the  disease.  Microblasts  have 
much  the  same  significance  as  normoblasts,  but  are 
less  common. 

Cabot's  ring  bodies  are  ring-  or  figure-of-8  shaped 
structures  which  have  been  observed  in  certain  of  the 
red  cells  in  pernicious  anemia,  lead-poisoning,  and  lym- 
phatic leukemia.  They  stain  red  with  Wright's  stain. 
Their  nature  is  unknown. 

2.  The  Leukocytes.~An  estimation  of  the  number  or 
percentage  of  each  variety  of  leukocyte  in  the  blood  is 
called  a  dijjerential  count.  It  probably  yields  more 
helpful  information  than  any  other  single  procedure  in 
blood  examinations. 

The  differential  count  is  best  made  upon  a  film  stained 
with  Wright's,  Harlow's,  or  Ehrlich's  stain.  Go  care- 
fully over  the  film  with  an  oil-immersion  lens,  using  a 
mechanical  stage  if  available.     Classify  each  leukocyte 


STUDY   OF  STAINED  BLOOD  23 1 

seen,  and  calculate  what  percentage  each  variety  is  of 
the  whole  number  classified.  For  accuracy,  50x3  to  1000 
leukocytes  must  be  classified;  for  approximate  results, 
300  are  sufficient.  Track  of  the  count  may  be  kept  by 
placing  a  mark  for  each  leukocyte  in  its  appropriate 
column,  ruled  upon  paper.  Some  workers  divide  a  slide- 
box  into  compartments  with  slides,  one  for  each  variety  of 
leukocyte,  and  drop  a  coffee-bean  into  the  appropriate 
compartment  when  a  cell  is  classified.  When  a  conve- 
nient number  of  coffee-beans  is  used  (any  multiple  of 
100),  the  percentage  calculation  is  extremely  easy. 

The  actual  number  of  each  variety  in  a  cubic  milU- 
meter  of  blood  is  easily  calculated  from  these  percentages, 
and  the  total  leukocyte  count.  An  increase  in  actual 
number  is  an  absolute  increase;  an  increase  in  percentage 
only,  a  relative  increase.  It  is  evident  that  an  absolute 
increase  of  any  variety  may  be  accompanied  by  a  relative 
decrease. 

A  record  is  generally  kept  of  the  number  of  nucleated 
red  cells  seen  during  a  differential  count  of  leukocytes. 

The  usual  classification  of  leukocytes  is  based  upon 
their  size,  their  nuclei,  and  the  staining  properties  of  the 
granules  which  many  of  them  contain.  It  is  not  alto- 
gether satisfactory,  but  is  probably  the  best  which  our 
present  knowledge  permits. 

The  writer  has  foimd  the  table  (Fig.  88,  p.  232) 
very  helpful  in  impressing  this  classification  upon  the 
student.  It  makes  no  attempt  to  indicate  histogenetic 
Telationships.  The  leukocytes  of  normal  blood  fall  into 
two  groups,  each  including  three  types.  The  cells  in 
Group  I  contain  single,  round,  oval  or  horseshoe-shaped 
nuclei,  and  have  few  or  no  granules  in  their  cytoplasm. 


232 


THE   BLOOD 


The  stippling  of  the  cytoplasm  shown  in  the  diagram 
represents  the  finely  granular  appearance  of  protoplasiri, 
not  true  granulation.  The  cells  in  Group  II  are  polymor- 
phonuclear and  contain  granules  which  are  distinguished 
by  their  size  and  staining  reactions.  In  its  structure  the 
chief  abnormal  leukocyte,  the  myelocyte,  combines  the 

LEUKOCYTES 

NORMAL  /^DNORM^L 


MONONUCLE/7R  ~ 
NON  -  GR/^NUL/Jr? 

1-LYMPHOCVTE       20-30°/„ 
2-L/5RGE  MONONUCLE^RI 

3  Tr?/1N5ITI0N^L  J 

POLYMORPHONUCLEAR 
GR/7NUL/7R  


MyELOOTE 


1-NEUTROPHIUCf 


2- EOSINOPHILIC  iM 


3-B-^SOPHILIC 


1-  ISIEUTPOPHILIC     60-75%  § 


2  -  EOSINOPHILIC 


2-4yo5i 


.'.-is-A 


3- BASOPHILIC  0.3%    BfflJ 

Fig.  88. — Outline  of  the  classification  of  leukocytes. 

two  groups,  being  mononuclear  like  Group  I,  and  gran- 
ular like  Group  II. 

(i)  Normal  Varieties. — {a)  Lymphocjrtes. — These  are 
small  mononuclear  cells  without  granules  (Plates  VI  and 
X).  They  are  about  the  size  of  a  red  corpuscle  or 
slightly  larger  (6-io  (u),  and  consist  of  a  single,  sharply 


10   HOITAMAJ^XH 

I    oriT      .t?.i;li\oT  >tn;    l)iixi    Igiildolfi^oril    ,^     :f:u'il>ijn    f^-jjiiludol 
Ifitne  bnfi  ^gisl   .  vl   ,j  ;9l'>eoqio^  b^  a  a<  .no 


'^   3no   ,b3ii;lfjui    ^m 

ifiluganJ  MJi  io  baeo.j 

inaiHSnoi  ^t{ioiu^i  ^B^hunpaom  ^3^^Bl  ,0j  ;  jhun'to 

i)  ni  e3g£t2  luol  ,^i  ;BnBlcrti  nEJnal  !o  •«i.  ■  -"AunBTg 

h  313W  rfJnuol  bnB  bnonv.  ■srf)   :3ji?.Biiiq  iBhslum  ndJTJJ  adJ  lo  abya 

■  no*  bsi  ,8i  ;nBiJi3)  ^Mitoh  Ho  ^bt  b  moil  n9>!«t  gbil?  ^mfw  ^Ht  moil 

I    .gnikjqiJa  l/ihi.'  '  nn-j 

'iin()iw  armol  ji  ;ub 


istingui^l 
'nicture  the 
"inbines  the 


Explanation  of  Plate  VI 

Stained  with  Wright's  stain.  Ail  drawn  to  same  scale. 
I,  Norma!  red  corpuscle  for  comparison;  2,  normoblasts,  one  with 
lohulatcd  nucleus;  3,  megaioblast  and  microblast.  The  megaloblast 
shows  a  considerable  degree  of  polychromatophilia;  4,  blood-plaques, 
one  lying  uy.on  a  red  corpuscle;  5,  lymphocytes,  large  and  small;  6, 
large  mononuclear  leukocyte;  7,  transitional  leukocyte;-  8,  polymor- 
phonuclear neutrophilic  leukocytes;  9,  eosinophilic  leukocytes,  one 
ruptured;  10,  basophilic  leukocyte;  11,  neutrophilic  myelocyte.  The 
granules  are  sometimes  less  numerous  and  less  distinct  than  here  shown; 
12,  eosinophilic  mvelocvtes;  13,  basophilic  myelocyte;  14,  ''irritation" 
or  "stimulation"  form,  with  small  vacuoles;  15,  degenerated  leukocytes: 
two  polymorphonuclear  neutrophiles,  one  ruptured,  one  swojlen  and 
vacuolated;  and  a  "basket  cell"  composed  of  an  irregular  meshwork 
of  nuclear  material;  16,  large  mononuclear  leukocyte  containing  f)igment- 
granules;  from  a  case  of  tertian  malaria;  17,  four  stages  in  the  asexuial 
cycle  of  the  tertian  malarial  parasite:  the  second  and  fourth  were  drawn 
from  the  same  slide  taken  from  a  case  of  double  tertian;  18,  red  corpuscle 
containing  tertian  parasite  and  showing  malarial  stippling;  19,  estivo- 
autumnal  malarial  parasites:  two  small  ring  forms  within  the  same 
red  cell,  and  a  crescent  with  remains  of  the  red  corpuscle  in  its  concavity. 


and  gran- 

"Dicse  are 
\  I  and 


PLATE  VI 


-•^'■-'   3 


4 


•      4    a 


"3»r 


10 


12 


13 


14 


16 


15 


17 


r*^ 


18 


G- 


19 


STUDY  OF  STAINED  BLOOD 


233 


defined,  deeply  staining  nucleus,  surrounded  by  a  narrow 
rim  of  protoplasm.  The  nucleus  is  generally  round,  but 
is  sometimes  indented  at  one  side.  Wright's  stain  gives 
the  nucleus  a  deep  purple  color  and  the  cytoplasm  a  pale 
robin's-egg  blue  in  typical  cells.  Larger  forms  of  lymph- 
ocytes are  frequently  found,  especially  in  the  blood  of 
children,  and  are  difficult  to  distinguish  from  the  large 
mononuclear  leukocytes.     It  is  possible  that  the  larger 


Fig.  89. — Lymphocytt).sis,  c.ise  of  pertussis  (  X 1000)  (courtesy  of  Dr.  W.  P.  Harlow). 


forms  are  young  lymphocytes,  which  become  smaller 
as  they  grow  older. 

Lymphocytes  are  formed  in  the  lymphoid  tissues, 
including  that  of  the  bone-marrow.  They  constitute, 
normally,  20  to  30  per  cent,  of  all  leukocytes,  or  about 
1000  to  3000  per  c.mm.  of  blood.  They  are  more  abun- 
dant in  the  blood  of  children. 

The  percentage  of  lymphocytes  is  usually  moderately 
increased  in  those  conditions  which  give  leukopenia, 


234  'i'llE    BLOOD 

especially  typhoid  fe\'er,  chlorosis,  pernicious  anemia, 
and  many  debilitated  conditions.  A  marked  increase, 
accompanied  by  an  increase  in  the  total  leukocyte  count, 
is  seen  in  pertussis  (Fig.  89)  and  lymphatic  leukemia.  In 
the  latter  the  lymphocytes  sometimes  exceed  98  per  cent. 
E.xophthalmic  goiter  commonly  gives  a  marked  relative 
lymphoc}tosis,  while  simple  goiter  does  not  affect  the 
l}mphocytes. 

(h)  Large  Mononuclear  Leukocjrtes  (Plate  VI). — 
These  cells  are  two  or  three  times  the  diameter  of  the 
normal  red  corpuscle.  Each  contains  a  single  round  or 
oval  nucleus,  often  located  eccentrically.  The  zone  of 
protoplasm  surrounding  the  nucleus  is  relatively  wide. 
With  Wright's  stain  the  nucleus  is  less  deeply  colored 
than  that  of  the  lymphocyte,  while  the  cytoplasm  is  very 
pale  blue  or  colorless,  and  sometimes  contains  a  few  red- 
dish granules.  The  size  of  the  cell,  the  width  of  the  zone 
of  cytoplasm,  and  the  depth  of  color  of  the  nucleus  are 
the  points  to  be  considered  in  distinguishing  between 
large  mononuclears  and  lymphocytes.  When  large 
forms  of  the  lymphocyte  are  present,  the  distinction  is 
often  difficult  or  impossible.  It  is  then  advisable  to 
count  the  two  cells  together  as  lymphocytes.  Indeed, 
they  are  regarded  by  some  hematologists  as  identical. 

Large  mononuclear  leukocytes  probably  originate  in 
the  bone-marrow  or  spleen.  Some  hold  that  they  are 
developed  from  the  endothelial  cells  of  the  blood-vessels. 
They  constitute  2  to  4  per  cent,  of  the  total  number  of 
leukocytes:  100  to  400  per  c.mm.  of  blood.  An  increase 
is  unusual  except  in  malaria,  where  it  is  quite  constantly 
observed,  and  where  many  of  the  cells  contain  engulfed 
pigment. 


STUDY  OF  STAINED  BLOOD 


235 


{c)  Transitional  Leukocytes  (Plate  VI). — These  are 
essentially  large  mononuclears  with  deeply  indented  or 
horseshoe-shaped  nuclei.  A  few  fine  neutrophilic  gran- 
ules are  sometimes  present  in  their  cytoplasm. 

They  are  probably  formed  from  the  large  mononuclears, 
and  occur  in  the  blood  in  about  the  same  numbers.  The 
two  cells  are  usually  counted  together,  constituting  4  to 
8  per  cent,  of  the  leukocytes. 


Fig.  90.— Marked  polymorphonuclear   neutrophilic  leukocytosis  ;  ,s  loooj   (courtesy  of 
Dr.  W.  P.  Harlow). 

{d)  Polymorphonuclear  Neutrophilic  Leukocytes  (Plate 
VI). — There  is  usually  no  difficulty  in  recognizing 
"these  cells.  Their  average  size  is  somewhat  less  than 
that  of  the  large  mononuclears.  The  nucleus  stains 
rather  deeply,  and  is  extremely  irregular,  often  assuming 
shapes  comparable  to  letters  of  the  alphabet,  E,  Z,  S,  etc. 


236  THE   BLOOD 

(Fig.  90).  Frequently  there  appear  to  be  several  separ- 
ate nuclei,  hence  the  widely  used  name,  "polynuclear  leu- 
kocyte." Upon  careful  inspection,  however,  delicate 
nuclear  bands  connecting  the  parts  can  usually  be  seen. 
The  cytoplasm  is  relatively  abundant,  and  contains 
great  numbers  of  very  fine  neutrophilic  granules  (Fig. 
93).  With  Wright's  stain  the  nucleus  is  bluish  purple, 
and  the  granules  reddish  lilac. 

Polymorphonuclear  leukocytes  are  formed  in  the  bone- 
marrow  from  neutrophilic  myelocytes.  They  constitute 
60  to  75  per  cent,  of  all  the  leukocytes:  3000  to  7500  per 
c.mm.  of  blood.  Increase  in  their  number  practically 
always  produces  an  increase  in  the  total  leukocyte  count, 
and  has  already  been  discussed  under  Polymorphonu- 
clear Leukocytosis.  The  leukocytes  of  pus,  pus-corpuscles, 
belong  almost  wholly  to  this  variety. 

A  comparison  of  the  percentage  of  polymorphonuclear 
cells  with  the  total  leukocyte  count  yields  more  informa- 
tion than  a  consideration  of  either  alone.  In  a  general 
way  the  percentage  represents  the  severity  of  the  infec- 
tion, or,  more  correctly,  the  degree  of  toxic  absorption; 
while  the  total  count  indicates  the  patient's  power  of 
resistance.  With  moderate  infection  and  good  resisting 
powers  the  leukocyte  count  and  the  percentage  of  poly- 
morphonuclears are  increased  proportionately.  When 
the  polymorphonuclear  percentage  is  increased  to  a  nota- 
bly greater  extent  than  is  the  total  number  of  leuko- 
cytes, no  matter  how  low  the  count,  either  very  poor 
resistance  or  a  very  severe  infection  may  be  inferred. 

Gibson  has  suggested  the  use  of  a  chart  to  express 
this  relationship  graphically  (Fig.  91).  Its  arrangement 
is  purely  arbitrary,  but  it  may  be  found  helpful  in  inter- 


STUDY  OF  STAINED  BLOOD 


237 


preting  counts.     An  ascending  line  from  left  to  right 
indicates  an  unfavorable  prognosis  in  proportion  as  the 


."^o  non 

/ 

<=)-'^ 

P-.'i  000 

/ 

on 

7>.o,ono 

/ 

RA 

1.*;  000 

_  / 

flO 

10,  000 

/ 

— 

■7-5 

, 

/ 

z 

.•^  000 

— 

7T> 



— 

Total  leuko- 
cyte count. 


Percentage  of 
polymorpho- 
nuclears. 


Fig.  91. — Gibson  chart  with  blood-count  in  two  cases  of  appendicitis:  Dotted  line  rep- 
resenting a  mild  case  with  prompt  recovery;  the  continuous  line,  a  very  virulent  strepto- 
coccic case  with  poor  resistance,  fKjritonitis,  and  early  death. 


line  approaches  the  vertical.  All  fatal  cases  show  a  ris- 
ing line.  A  descending  or  horizontal  line  suggests  a  very 
favorable  prognosis. 


238  THE   BLOOD 

It  is  a  matter  of  observation  that  in  the  absence  of 
acute  infectious  disease  or  of  inflammation  directly  in 
the  blood-stream  {e.  g.,  phlebitis,  sigmoid  sinusitis,  septic 
endocarditis),  a  polymorphonuclear  percentage  of  85  or 
over  points  very  strongly  to  gangrene  or  pus-formation 
somewhere  in  the  body.  On  the  other  hand,  excepting 
in  children,  where  the  percentage  is  normally  low,  pus 
is  uncommon  with  less  than  80  per  cent,  of  polymorpho- 
nuclears. 

Normally,  the  cytoplasm  of  leukocytes  stains  pale 
yellow  with  iodin.  Under  certain  pathologic  conditions 
the  cytoplasm  of  many  of  the  polymorphonuclears  stains 
diffusely  brown,  or  contains  granules  which  stain  reddish 
brown  with  iodin.  This  is  called  iodophilia.  Extracellu- 
lar iodin-staining  granules,  which  are  present  normally, 
are  more  numerous  in  iodophilia. 

This  iodin  reaction  occurs  in  all  purulent  conditions 
except  abscesses  which  are  thoroughly  walled  off  and 
purely  tuberculous  abscesses.  It  is  of  some  value  in 
diagnosis  between  serous  effusions  and  purulent  exudates, 
between  catarrhal  and  suppurative  processes  in  the  ap- 
pendix and  Fallopian  tube,  etc.  Its  importance,  how- 
ever, as  a  diagnostic  sign  of  suppuration  has  been  much 
exaggerated,  since  it  may  occur  in  any  general  toxemia, 
such  as  pneumonia,  influenza,  malignant  disease,  and 
puerperal  sepsis. 

To  demonstrate  iodophilia,  place  the  air-dried  films  in 
a  stoppered  bottle  containing  a  few  crystals  of  iodin  until 
they  become  yellow.  Mount  in  syrup  of  levulose  and 
examine  with  an  immersion  objective. 

Arneth  classifies  pohonorphonuclear  leukocytes  into 
five  groups,  according  to  the  number  of  lobes  which  the 


STUDY  OF  STAINED  BLOOD  239 

nucleus  shows.  The  percentage  of  cells  in  each  group 
is  fairly  constant  in  health,  but  shows  considerable  varia- 
tion in  disease. 

(e)  Eosinophilic  Leukocjrtes,  or  "  Eosinophiles " 
(Plate  VI). — The  structure  of  these  cells  is  similar  to  that 
of  the  polymorphonuclear  neutrophiles,  with  the  striking 
difference  that,  instead  of  fine  neutrophilic  granules,  their 
cytoplasm  contains  coarse,  round  or  oval  granules  having 
a  strong  affinity  for  acid  stains.  They  are  easily  recog- 
nized by  the  size  and  color  of  the  granules,  which  stain 
bright  red  with  Wright's  stain  (Fig.  93).  Their  cyto- 
plasm has  generally  a  faint  sky-blue  tinge,  and  the  nu- 
cleus stains  somewhat  less  deeply  than  that  of  the 
polymorphonuclear  neutrophile. 

Eosinophiles  are  formed  in  the  bone-marrow  from 
eosinophilic  myelocytes.  Their  normal  number  varies 
from  50  to  400  per  c.mm.  of  blood,  or  i  to  4  per  cent,  of 
the  leukocytes.  An  increase  is  called  eosinophilia,  and  is 
better  determined  by  the  actual  number  than  by  the 
percentage. 

Slight  eosinophilia  is  physiologic  during  menstruation. 
Marked  eosinophilia  is  always  pathologic.  It  occurs  in 
a  variety  of  conditions,  the  most  important  of  which  are : 
infection  by  animal  parasites;  bronchial  asthma;  myeloge- 
nous leukemia;  scarlet  fever,  and  many  skin  diseases. 

{a)  Eosinophilia  may  be  a  symptom  of  infection  by  any 
of  the  worms.  It  is  fairly  constant  in  trichinosis,  uncinaria- 
sis, filariasis,  and  echinococcus  disease.  In  this  country 
an  unexplained  marked  eosinophilia  warrants  examina- 
tion of  a  portion  of  muscle  for  Trichinella  spiralis  (p.  363). 

{h)  True  bronchial  asthma  commonly  gives  a  marked 
eosinophilia  during  and  following  the  paroxysms.     This 


240  THE   BLOOD 

is  helpful  in  excluding  asthma  of  other  origin.  Eosino- 
philes  also  appear  in  the  sputum  in  large  numbers. 

(c)  In  myelogenous  leukemia  there  is  almost  invariably 
an  absolute  increase  of  eosinophiles,  although,  owing  to 
the  great  increase  of  other  leukocytes,  the  percentage  is 
usually  diminished.  Dwarf  and  giant  forms  are  often 
numerous. 

id)  Scarlet  fever  is  frequently  accompanied  by  eosino- 
philia,  which  may  help  to  distinguish  it  from  measles. 

i^::       ^  m_^^>^     \-\  ^^ --i 

Fig.  92. — Basophilic  leukocytes.    .\\.  the  right  is  also  a  normoblast  undergoing  mitosis 
( X  1000)  (photographs  by  the  author). 

(e)  Eosinophilia  has  been  observed  in  a  large  number 
of  skin  diseases,  notably  pemphigus,  prurigo,  psoriasis, 
and  urticaria.  It  probably  depends  less  upon  the  vari- 
ety of  the  disease  than  upon  its  extent. 

(/)  Basophilic  Leukocytes  or  *'  Mast-cells  "  (Plate 
VI). — In  general,  these  resemble  polymorphonuclear  neu- 
trophiles  except  that  the  nucleus  is  less  irregular  and  that 
the  granules  are  larger  and  have  a  strong  affinity  for 
basic  stains.  They  are  easily  recognized  (Figs.  92  and  93). 
With  Wright's  stain  the  granules  are  deep  purple,  while 


STUDY  OF  STAINED  BLOOD  24 1 

the  nucleus  is  pale  blue  and  is  often  nearly  or  quite  hid- 
den by  the  granules,  so  that  its  form  is  difficult  to  make 
out.  These  granules  are  not  colored  by  Ehrlich's  stain. 
The  nature  of  mast-cells  is  undetermined.  They 
probably  originate  in  the  bone-marrow.  They  are  least 
numerous  of  the  leukocytes  in  normal  blood,  rarely  ex- 
ceeding 0.5  per  cent.,  or  25  to  50  per  c.mm.  A  notable 
increase  is  Umited  almost  exclusively  to  myelogenous 
leukemia,  where  they  are  sometimes  very  numerous. 


B   .      . 

Fig.  93. — Ruptured  leukocytes,  showing  relative  size  of  granules:  A,  neutrophilic;   B, 
eosinophilic;  C,  basophilic  (X  looo)  (photographs  by  the  author). 

(2)  Abnormal  Varieties. — (a)  Myelocytes  (Plate  VI  and 
Fig.  94). — These  are  large  mononuclear  cells  whose  cyto- 
plasm is  filled  with  granules.  Typically,  the  nucleus  occu- 
pies about  one-half  of  the  cell,  and  is  round  or  oval.  It  is 
sometimes  indented,  with  its  convex  side  in  contact  with 
the  periphery  of  the  cell.  It  stains  rather  feebly.  The 
average  diameter  of  this  cell  (about  15.75  !^)  is  greater 
than  that  of  any  other  leukocyte,  but  there  is  much  varia- 
tion in  size  among  individual  cells.  Myelocytes  are 
named  according  to  the  character  of  their  granules — 
neutrophilic,  eosinophilic,  and  basophilic  myelocytes. 
These  granules  are  identical  with   the   corresponding 

16 


242  THE  BLOOD 

granules  in  the  leukocytes  just  described.     The  occur- 
rence of  two  kinds  of  granules  in  the  same  cell  is  rare. 

Myelocytes  are  the  bone-marrow  cells  from  which  the 
corresponding  granular  leukocytes  are  developed.  Their 
presence  in  the  blood  in  considerable  numbers  is  diagnos- 
tic of  myelogenous  leukemia.  The  neutrophilic  form  is 
the  least  significant.  A  few  of  these  may  be  present  in 
very  marked  leukocytosis  or  any  severe  blood  condition, 
as  pernicious  anemia.     Eosinophilic  myelocytes  are  found 


A  B 

Fig.  94. — Myelocytes  from  blood  of  myelogenous  leukemia:  A,  Neutrophilic;  B,  eosino- 
philic (X  1000)  (photographs  by  the  author). 

only  in  myelogenous  leukemia,  where  they  are  often  very 
numerous.  The  basophilic  variety  is  less  common,  and 
is  confined  to  long-standing,  severe  myelogenous  leu- 
kemia. 

(b)  Atypic  Forms. — Leukocytes  which  do  not  fit  in 
with  the  above  classification  are  not  infrequently  met, 
especially  in  high-grade  leukocytosis,  pernicious  anemia, 
and  leukemia.  The  nature  of  most  of  them  is  not  clear, 
and  their  number  is  usually  so  small  that  they  may  be 


STUDY  OF  STAINED  BLOOD  243 

disregarded  in  making  a  differential  count.    Among  them 
are: 

(a)  Border-line  forms  between  polymorphonuclear 
neutrophils  and  neutrophilic  myelocytes. 

(b)  Small  neutrophilic  cells  with  a  single  round, 
deeply  staining  nucleus;  they  probably  result  from  di- 
vision of  polymorphonuclear  neutrophiles. 

(c)  "  Irritation  forms  " — large  non-granular  mono- 
nuclear cells,  whose  cytoplasm  stains  fairly  deep  purple 
with  Wright's  stain,  and  intense  brown  with  Ehrlich's: 


Fig.  9S- — A  clu^U;r  of  blood-plaques  and  two  plaques  lying  upon  a  red  cell  and  simu- 
lating malarial  parasites  ( X  1000)  (photograph  by  the  author). 

they  appear  in  the  blood  under  the  same  conditions  as 
myelocytes. 

(d)  Degenerated  forms:  vacuolated  leukocytes,  or 
merely  palely  or  deeply  staining  homogeneous  or  retic- 
ulated masses  of  chromatin  (the  so-called  "  basket-cells," 
Plate  VI). 

■  3.  Blood=plaques.— These  are  not  colored  by  Ehrlich's 
stain  nor  by  eosin  and  methylene-blue.  With  Wright's 
stain  they  appear  as  spheric  or  ovoid,  reddish  to  violet, 
granular  bodies,  2  to  4  ^  in  diameter.     When  well  stained 


244  THE    BLOOD 

a  delicate  hyaline  peripheral  zone  can  be  distinguished. 
In  ordinary  blood-smears  they  are  usually  clumped  in 
masses.  A  single  platelet  lying  upon  a  red  corpuscle  may 
easily  be  mistaken  for  a  malarial  parasite  (Plate  VI  and 

Fig.  95)- 

Blood-platelets  are  being  much  studied  at  present,  but, 
aside  from  the  facts  mentioned  under  their  enumeration 
(p.  213),  little  of  clinical  value  has  been  learned.  They 
have  been  variously  regarded  as  very  young  red  corpus- 
cles (the  "  hematoblasts"  of  Hayem),  as  disintegration 
products  of  leukocytes,  as  remnants  of  extruded  nuclei 
of  erythrocytes,  and  as  independent  nucleated  bodies. 
The  most  probable  explanation  of  their  origin  seems  to 
be  that  of  J.  H.  Wright,  who,  from  his  recent  studies, 
regards  them  as  detached  portions  of  the  cytoplasm  of 
certain  giant-cells  of  the  bone-marrow  and  spleen. 

VIII.    BLOOD  PARASITES 
A.   Bacteria 

Bacteriologic  study  of  the  blood  is  useful  in  many 
conditions,  but  in  general,  the  elaborate  technic  involved 
takes  it  out  of  the  reach  of  the  clinician.  As  applied  to 
the  diagnosis  of  t\T3hoid  fever,  however,  the  technic  of 
blood-cultures  has  been  so  simplified  that  it  can  be  car- 
ried through  by  any  one  who  is  competent  to  do  the 
simplest  cultural  work. 

Typhoid  bacilli  can  be  detected  in  the  blood  in  prac- 
tically every  case  of  typhoid  fever  in  the  first  week  of  the 
disease;  in  about  80  to  85  per  cent,  of  cases  in  the  second 
week;  and  in  decreasing  percentages  in  the  later  weeks. 
The  blood-culture,  therefore,  offers  the  most  certain  means 


BLOOD  PARASITES  245 

of  early  diagnosis.  It  is  in  a  sense  complementary  to  the 
Widal  reaction,  the  former  decreasing  and  the  latter 
increasing  in  reliability  as  the  disease  progresses.  The 
blood-culture  gives  best  results  before  the  Widal  appears, 
as  one  Would  expect  from  the  fact  that  the  Widal  test 
depends  upon  the  presence  of  antibodies  which  destroy, 
or,  at  least  injure,  the  bacilli.  The  two  methods  to- 
gether will  establish  the  diagnosis  in  practically  every 
case  at  any  stage.  Bacilli  disappear  from  the  blood  in 
convalescence  and  reappear  in  a  relapse. 

Technic  of  Blood-Cultures  in  Typhoid  Fever. — The  blood 
may  be  obtained  in  one  of  two  ways: 

(a)  With  a  spring-lancet  make  a  deep  puncture  in  the 
edge  (not  the  side)  of  the  lobe  of  the  ear,  as  for  a  blood-count. 
Allow  the  blood  to  drop  directly  into  a  short  culture-tube 
containing  the  bile  medium.  By  gentle  milking,  20  to  40 
drops  can  usually  be  obtained.  This  simple  method  of  ob- 
taining blood  is  especially  applicable  during  the  first  week  of 
the  disease  when  bacilli  are  abundant.  Contamination  with 
skin  cocci  is  possible,  but  does  not  usually  interfere  when  the 
bile  medium  is  used. 

(b)  In  the  later  weeks  of  the  disease  a  larger  quantity  of 
blood  is  needed.  Prepare  the  skin  on  the  front  of  the  elbow, 
as  for  a  minor  operation,  or  simply  rub  well  with  alcohol. 
Tie  a  bandage  tightly  aroimd  the  upper  arm,  have  the  patient 
open  and  close  the  fist  a  few  times,  and  when  the  veins  are 
sufiiciently  distended  insert  a  hypodermic  needle  attached 
to  a  syringe  into  any  vein  that  is  prominent.  The  needle 
should  go  through  the  skin  about  J  inch  from  the  vein  with 
the  bevel  at  its  tip  uppermost,  and  should  enter  the  vein 
from  the  side  in  a  direction  opposite  to  the  blood-current  (Fig. 
96).  Unless  too  small  a  needle  is  used,  blood  will  begin  to 
rise  in  the  syringe  as  soon  as  the  needle  has  entered  the  vein. 


246 


THE   BLOOD 


Suction  is  not  necessary.  When  sufficient  blood  is  obtained, 
the  bandage  is  first  removed,  the  needle  is  withdrawn,  and 
the  blood  is  allowed  to  run  into  a  tube  of  culture-medium. 
It  is  usually  easy  to  secure  5  to  10  c.c.  of  blood.  The  proced- 
ure causes  the  patient  surprisingly  little  inconvenience,  sel- 
dom more  than  does  an  ordinary  hyj^odermic  injection. 
There  is  rarely  any  difficulty  in  entering  the  vein  except  in 
children,  and  in  adults  when  the  arm  is  fat  and  the  veins  are 
small.  If  desired,  one  of  the  veins  about  the  ankle  can  be 
used.     Instead  of  a  syringe  one  can  use  a  large  glass  tube 


Fig.  q6. — Method  of  obtaining  blood  for  a  blood-culture. 


which  has  been  drawn  out  at  the  ends  and  one  end  ground  to 
fit  a  "  slip-on  "  needle.  Either  a  large  hypodermic  needle  or 
a  small  antitoxin  needle  may  be  used.  These  little  instru- 
ments (Fig.  96)  can  be  made  by  any  glass-blower  at  a 
cost  of  about  fifty  cents,  and  several  of  them  can  be  kept 
on  hand  in  test-tubes  sterilized  ready  for  use. 

As  special  culture-medium,  ox-bile  is  generally  used.  It 
favors  the  growth  of  the  typhoid  bacillus  and  retards  the 
growth  of  other  organisms.    A  good  formula  is  given  on  p.  405. 

As  soon  as  convenient  after  the  blood  is  added,  place  the 
tubes  in  the  incubator.     After  about  twelve  hours  examine 


BLOOD  PARASITES  247 

for  motile  bacilli.  If  none  are  found,  transfer  a  few  drops 
to  tubes  of  bouillon  or  solidified  blood-serum  and  incubate  for 
twelve  hours  longer.  If  motile,  Gram-negative  bacilli  are 
foimd;  they  are  almost  certainly  typhoid  bacilli.  Further 
study  is  not  necessary  in  practice,  although  desirable  from 
a  scientific  point  of  view.  The  only  bacilli  which  might  cause 
confusion  are  the  paratyphoid  and  colon  bacilli,  which  can 
be  distinguished  by  gas  production  in  glucose  media,  indol 
production,  and  their  effect  upon  litmus  milk.  The  agglutin- 
ation test  for  the  identity  of  the  bacillus  is  not  available 
clinically,  since  freshly  isolated  bacilli  do  not  agglutinate 
well. 

B.   Animal  Parasites 

Of  the  animal  parasites  which  have  been  found  in  the 
blood,  five  are  interesting  clinically:  the  spirochaeta  of 
relapsing  fever;  trypanosomes;  malarial  parasites;  filarial 
embryos;  and  the  embryos  of  Trichinella  spiralis. 

1.  Spirochaeta  recurrentis  is  described  on  p.  330. 

2.  Trypanosoma  Qambiense. — Various  trypanosomes 
are  common  in  the  blood  of  fishes,  amphibians,  birds,  and 
mammals  (Fig.  113).  They  live  in  the  blood-plasma  and 
do  not  attack  the  corpuscles.  In  some  animals  they  are 
apparently  harmless;  in  others  they  are  an  important 
cause  of  disease.    They  are  discussed  more  fully  on  p.  333. 

The  trypanosome  of  human  blood,  Trypanosoma  gam- 
biense  (Plate  VII),  is  an  actively  motile,  spindle-shaped 
organism,  two  or  three  times  the  diameter  of  a  red  cor- 
puscle in  length,  with  an  undulating  membrane  which 
terminates  at  the  anterior  end  in  a  long  ilagellum.  It  can 
be  seen  with  medium  power  objectives  in  fresh  blood,  but 
is  best  studied  with  an  oil-immersion  lens  in  preparations 
stained  as  for  the  malarial  parasite.     Human  trypano- 


248  THE   BLOOD 

somiasis  is  common  in  Africa.  As  a  rule,  it  is  a  very 
chronic  disease.  "  Sleeping  sickness  "  is  a  late  stage  when 
the  organisms  have  invaded  the  cerebrospinal  fluid. 
Infection  is  carried  by  the  tsetse  fly,  Glossina  palpalis. 

3.  The  Malarial  Parasites.— These  protozoa  belong  to 
the  Sporozoa  (p.  338),  order  Hemosporidia,  the  mem- 
bers of  which  are  parasites  in  the  blood  of  a  great 
variety  of  vertebrates.  Three  species,  constituting  the 
genus  Plasmodium,  are  associated  with  malarial  fever  in 
man :  Plasmodium  vivax,  P.  malarice,  and  P.  falciparum, 
the  parasites  respectively  of  the  tertian,  quartan,  and 
estivo-autumnal  types  of  malaria.  The  life  histories  of 
the  three  are  so  similar  that  they  may  well  be  described 
together. 

(i)  Life  Histories. — There  are  two  cycles  of  develop- 
ment: one,  the  asexual,  in  the  blood  of  man;  and  the 
other,  the  sexual,  in  the  intestinal  tract  of  a  particular 
genus  of  mosquito,  AnopJieles. 

(a)  Asexual  Cycle. — The  young  organism  enters  the 
blood  through  the  bite  of  the  mosquito.  It  makes  its  way 
into  a  red  corpuscle,  where  it  appears  as  a  small,  pale 
"  hyaline  "  body.  This  body  exhibits  ameboid  movement 
and  increases  in  size.  Soon,  dark-brown  granules  derived 
from  the  hemoglobin  of  the  corpuscle  make  their  appear- 
ance within  it.  When  it  has  reached  its  full  size — filling 
and  distending  the  corpuscle  in  the  case  of  the  tertian 
parasite,  smaller  in  the  others — the  pigment  granules 
gather  at  the  center  or  at  one  side;  the  organism  divides 
into  a  number  of  small  hyaline  bodies,  the  spores  or 
merozoites;  and  the  red  corpuscle  bursts,  setting  spores 
and  pigment  free  in  the  blood-plasma.  This  is  called 
segmentation.     It  coincides  with,  and  by  liberation  of 


PLATE    VII 


Trypanosoma  gambiense  (slide  presented  by  Professor  F.  G.  Novy). 


E 

1 

V 

Tertian  malarial  parasites,  one  red  Estivo-autumnal  malarial  para- 

cell  showing  malarial  stippling.  sites,     small     ring     forms      and 

crescents. 


Spinu  h.cta  novyi. 
Animal  parasites  of  the  blood;   X  looo  (photographs  by  the  author). 


BLOOD  PARASITES  249 

toxins  causes,  the  paroxysm  of  the  disease.  A  consider- 
able number  of  the  spores  are  destroyed  by  leukocytes  or 
other  agencies;  the  remainder  enter  other  corpuscles 
and  repeat  the  cycle.  Many  of  the  pigment  granules 
are  taken  up  by  leukocytes'  In  estivo-autumnal  fever 
segmentation  occurs  in  the  internal  organs  and  the  seg- 
menting and  larger  pigmented  forms  are  not  seen  in  the 
peripheral  blood. 

The  asexual  cycle  of  the  tertian  organism  occupies 
forty-eight  hours;. of  the  quartan,  seventy- two  hours;  of 
the  estivo-autumnal,  an  indefinite  time — usually  twenty- 
four  to  forty-eight  hours. 

The  parasites  are  thus  present  in  the  blood  in  great 
groups,  all  the  individuals  of  which  reach  maturity  and 
segment  at  approximately  the  same  time.  This  explains 
the  regular  recurrence  of  the  paroxysms  at  intervals  cor- 
responding to  the  time  occupied  by  the  asexual  cycle  of 
the  parasite.  Not  infrequently  there  is  multiple  infection, 
one  group  reaching  maturity  while  the  others  are  still 
young;  but  the  presence  of  two  groups  which  segment 
upon  the  same  day  is  extremely  rare.  Fevers  of  longer 
intervals — six,  eight,  ten  days — are  probably  due  to  the 
ability  of  the  body,  sometimes  of  itself,  sometimes  by  aid 
of  quinin,  to  resist  the  parasites,  so  that  numbers  suffi- 
cient to  cause  a  paroxysm  do  not  accumulate  in  the  blood 
until  after  several  repetitions  of  the  asexual  cycle.  In 
estivo-autumnal  fever  the  regular  grouping,  while  usually 
present  at  first,  is  soon  lost,  thus  causing  "irregular 
malaria." 

(b)  Sexual  Cycle. — Besides  the  ameboid  individuals 
which  pass  through  the  asexual  cycle,  there  are  present 
with  them  in  the  blood  many  individuals  with  sexual 


250  THE   BLOOD 

properties.  These  are  called  gametes.  They  do  not 
undergo  segmentation,  but  grow  to  adult  size  and  remain 
inactive  in  the  blood  until  taken  up  by  a  mosquito. 
Many  of  them  are  apparently  extracellular,  but  stained 
preparations  usually  show  them  to  be  surrounded  by  the 
remains  of  a  corpuscle.  In  tertian  and  quartan  malaria 
they  cannot  easily  be  distinguished  from  the  asex- 
ual individuals  until  a  variable  time  after  the  blood 
leaves  the  body,  when  the  male  gamete  sends  out 
one  or  more  flagella.  In  estivo-autumnal  malaria  the 
gametes  take  distinctive  ovoid  and  crescentic  forms,  and 
are  not  difficult  to  recognize.  They  are  very  resistant  to 
quinin  and  often  persist  in  the  blood  long  after  the 
ameboid  forms  have  been  destroyed,  but  are  probably 
incapable  of  continuing  the  disease  until  they  have  passed 
through  the  cycle  in  the  mosquito. 

When  a  malarious  person  is  bitten  by  a  mosquito,  the 
gametes  are  taken  with  the  blood  into  its  stomach.  Here 
a  flagellum  from  the  male  unites  with  the  female,  which 
soon  thereafter  becomes  encysted  in  the  wall  of  the  intes- 
tine. After  a  time  it  ruptures,  liberating  many  minute 
rods,  or  sporozoites,  which  have  formed  within  it.  These 
migrate  to  the  salivary  glands,  and  are  carried  into  the 
blood  of  the  person  whom  the  mosquito  bites.  Here  they 
enter  red  corpuscles  as  young  malarial  parasites,  and  the 
majority  pass  through  the  asexual  cycle  just  described. 

The  sexual  cycle  can  take  place  only  within  the  body 
of  one  genus  of  mosquito.  Anopheles.  Absence  of  this 
mosquito  from  certain  districts  explains  the  absence  of 
malaria.  It  is  distinguished  from  our  common  house- 
mosquito,  Culex,  by  the  relative  lengths  of  proboscis  and 
palpi  (Fig.  97),  which  can  be  seen  \\ith  a  hand-lens,  by 


BLOOD  PARASITES         '-^^flAf-.y  ^SJ 

r 

its  attitude  when  resting,  and  by  its  dappled  wing  (Pi^.f  /:  j 
98).  Anopheles  is  strictly  nocturnal  in  its  habits;  it 
usually  flies  low,  and  rarely  travels  more  than  a  few 
hundred  yards  from  its  breeding-place,  although  it  may 
be  carried  by  winds.  These  facts  explain  certain  peculi- 
arities in  malarial  infection;  thus,  infection  occurs  prac- 
tically only  at  night;  it  is  most  common  near  stagnant 
water,  especially  upon  the  side  toward  which  the  pre- 
vailing winds  blow;  and  the  danger  is  greater  when  per- 


Fig.  97. — Mosquitoes — Culex  (i)  and  Anopheles  (2)  (Bergey). 

sons  sleep  upon  or  near  the  ground  than  in  upper  stories 
of  buildings.  The  insects  frequently  hibernate  in  warmed 
houses,  and  may  bite  during  the  winter.  A  mosquito 
becomes  dangerous  in  eight  to  fourteen  days  after  it 
bites  a  malarious  person,  and  remains  so  throughout 
its  Hfe. 

(2)  Detection. — Search  for  the  malarial  parasite  may 
be  made  in  either  fresh  blood  or  stained  films.  If  possible, 
the  blood  should  be  obtained  a  few  hours  before  the  chiU 
— never  during  it  nor  within  a  few  hours  afterward,  since 


252 


r<\ 


THE  BLOOD 


at  that  time  (in  single  infections)  only  the  very  young, 
unpigmented  forms  are  present,  and  these  are  the  most 
difficult  to  find  and  recognize.  Sometimes  many  para- 
sites are  found  in  a  microscopic  field;  sometimes,  especi- 


Fig.  98. — Showing,  on  the  left.  Anopheles  in  resting  position,  its  dappled  wing,  and 
the  position  of  its  larvae  in  water;  on  the  right,  Culex  in  resting  position,  its  plain  wing, 
and  the  position  of  its  larvae  in  water.  The  arrows  indicate  the  directions  taken  by  the 
larvae  when  the  water  is  disturbed  (Abbott). 


ally  in  estivo-autumnal  infection,  owing  to  accumulation 
in  internal  organs,  careful  search  is  required  to  find  any, 
despite  very  severe  symptoms.  Quinin  causes  them 
rapidly  to  disappear  from  the  peripheral  blood,  and  few 
or  none  may  be  found  after  its  administration.     In  the 


BLOOD  PARASITES,        ~   '"'^/^/^/  ^ 

absence  of  organisms,  the  presence  of  pigment  granul6&f  A  f 
within  leukocytes — polymorphonuclears  and  large  mono-     ^. . 
nuclears — may  be   taken  as  presumptive   evidence  of 
malaria.     Pigmented  leukocytes   (Plate  VI)   are  most 
numerous  after  a  paroxysm. 

(a)  In  Fresh  Unstained  Blood  (Plate  VIII) . — Obtain  a 
small  drop  of  blood  from  the  finger  or  lobe  of  the  ear. 
Touch  the  center  of  a  cover-glass  to  the  top  of  the  drop 
and  quickly  place  it,  blood  side  down,  upon  a  slide.  If 
the  slide  and  cover  be  perfectly  clean  and  the  drop  not 
too  large,  the  blood  will  spread  out  so  as  to  present  only 
one  layer  of  corpuscles.  Search  with  an  oil-immersion 
objective,  using  very  subdued  light. 

The  young  organisms  appear  as  small,  round,  ring-like 
or  irregular,  colorless  bodies  within  red  corpuscles. .  The 
light  spots  caused  by  crenation  and  other  changes  in  the 
corpuscles  are  frequently  mistaken  for  them,  but  are 
generally  more  refractive  or  have  more  sharply  defined 
edges.  The  older  forms  are  larger  colorless  bodies  con- 
taining granules  of  brown  pigment.  In  the  case  of  the 
tertian  parasite,  these  granules  have  active  vibratory 
motion,  which  renders  them  conspicuous;  and  as  the 
parasite  itself  is  very  pale,  one  may  see  only  a  large  pale 
corpuscle  in  which  fine  pigment  granules  are  dancing. 
Segmenting  organisms,  when  typic,  appear  as  rosets, 
often  compared  to  daisies,  the  petals  of  which  represent 
the  segments,  while  the  central  brown  portion  represents 
the  pigment.  Tertian  segmenting  forms  are  less  fre- 
quently typic  than  quartan.  Flagellated  forms  are  not 
seen  until  ten  to  twenty  minutes  after  the  blood  has  left 
the  vessels.  As  Cabot  suggests,  one  should,  while  search- 
ing, keep  a  sharp  lookout  for  unusually  large  or  pale  cor- 


254  THE   BLOOD 

pu.sdes,  and  for  anything  which  is  brown  or  black  or  in 
motion. 

(b)  In  Stained  Films  (Plates  VI  and  VII). — Recogni- 
tion of  the  parasite,  especially  the  young  forms,  is  much 
easier  in  films  stained  by  Wright's  or  some  similar  stain 
than  in  fresh  blood.  When  very  scarce,  they  may  some- 
times be  found,  although  their  structure  is  not  well  shown 
b\-  the  method  of  Ruge.  This  consists  in  spreading  a  very 
thick  layer  of  blood,  drying,  placing  for  a  few  minutes  in 
a  fluid  containing  5  per  cent,  formalin  and  i  per  cent, 
acetic  acid,  which  removes  the  hemoglobin  and  fixes  the 
smear,  rinsing,  drying,  and  finally  staining.  Carbol- 
thionin  is  very  useful  for  this  purpose.  If  Wright's 
stain  be  used  in  this  method,  it  is  recommended  that  the 
preparation  be  subsequently  stained  for  a  half-minute 
with  borax-methylene-blue  (borax,  5 ;  methylene-blue,  2 ; 
water,  100). 

In  films  which  are  properly  stained  with  W^right's  fluid 
the  young  organisms  are  small,  round,  ring-like  or  irreg- 
ular, sky-blue  bodies,  each  with  a  very  small,  sharply  de- 
fined, reddish-purple  chromatin  mass.  Many  structures 
— deposits  of  stain,  dirt,  blood-plaques  lying  upon  red 
cells  (Fig.  95),  etc.-  -may  simulate  them,  but  should  not  de- 
ceive one  who  looks  carefully  for  both  the  blue  cytoplasm 
and  the  reddish-purple  chromatin.  A  plaque  upon  a  red 
corpuscle  is  surrounded  by  a  colorless  zone  rather  than  by 
a  distinct  blue  body.  Young  estivo-autumnal  parasites 
commonly  take  a  "  ring  "  form  (the  chromatin  mass  rep- 
resenting the  jewel),  which  is  infrequently  assumed  by 
the  other  varieties.  The  older  tertian  and  quartan  or- 
ganisms show  larger  sky-blue  bodies  with  more  reticular 
chromatin,  and  contain  brown  granules  of  pigment,  which, 


ITIV   3TAJT   ?0  VOlTA'AASHy.3 

J  JV/3H  bnij  wx^riT)  (baniBJano)  aaJiafiiBq  luhjiifirn  loennoi  ei;on£7 
II  ,3vraul)ni  .ri  n)   u   :»in».\rir.^-\t>  nxsiliaT  .wiKuhni  ,oi  oj  i 
•  '/ile')  .'jviaobni   .^e   oJ   8i    jarri' 
rfjiw   rmv>l  anilBvri   ,£   ;rmol  ■; 

■H--    •■•   .»     ■■   ,<-,    .'.,,...    ujjnarn:' "Tj-IIuI   ,^   ;fmol  h-))i"jrn 

.II  ;imoi  3jj3ll3S*fi  ,oi   ;rmol  :  iBlulhtentyo  .q- ;gunot 

igiq  bsqobvab  vllui  ,4,1   jarrnol  :  ^.  j  ,|r  1   ,£i   ;rrno]  amtevri 

in  ,0c  ,pi  ,81  ;fmo}  oifilJogfifi  ,71  ;8rmol  j^niJnamsag  ,t^i  ,gi  ;rtno1 
'^3^  rt-s  ,?;<:  ;grmo}  bajnamgiq  ,ss  .it  .armol  griilfivri  a>(il-2ao7o  bni; 

.aJna-ieoT)  ,;c  ,{>s  .js  ;8rmol 


Explanation  of  Plate  VIII 

Various  forms  of  malarial  parasites  (unstained)  (Thayer  and  Hewetson). 
I  to  lo,  inclusive,  Tertian  organisms;  ii  to  17,  inclusive,  quartan 
organisms;  18  to  27,  inclusive,  estivo-autumnal  organisms;  i,  young 
hyaline  form;  2,  hyaline  form  with  beginning  pigmentation;  3,  pig- 
mented form;  4,  full-grown  pigmented  form;  5,  6,  7,  8,  segmenting 
forms;  g,  extracellular  pigmented  form;  10,  flagellate  form;  11,  young 
hyaline  form;  12,  13,  pigmented  forms;  14,  fully  developed  pigmented 
form;  15,  16,  segmenting  forms;  17,  flagellate  form;  18,  19,  20,  ring-like 
and  cross-like  hyaline  forms;  21,  22,  pigmented  forms;  23,  24,  segmenting 
forms;  25,  26,  27,  crescents. 


PLATE  VIII 


3  4  5  6 


//  /?  /?  M 


r 


15  J6  17 


^i::^ 


^J< 


JS  19  20  2 J  22 


^i  24  25  26  27 


BLOOD  PARASITES  255 

however,  is  less  evident  than  in  the  living  parasite.  The 
chromatin  is  often  scattered  through  the  cytoplasm  or 
apparently  outside  of  it,  and  is  sometimes  difficult  to  see 
clearly.  Typical ''  segmenters  "  present  a  ring  of  rounded 
segments  or  spores,  each  with  a  small,  dot-like  chromatin 
mass.  With  the  tertian  parasite,  the  segments  more  fre- 
quently form  an  irregular  cluster.  The  pigment  is  col- 
lected near  the  center  or  scattered  among  the  segments. 
In  estivo-autumnal  fever  usually  only  the  small  "  ring 
bodies  "  and  the  crescentic  and  ovoid  gametes  are  seen 


^ 


Fig.  gg. — Filarial  embryos  in  blood.    Stained.    Red  corpuscles  decolorized;  a  few  leuko- 
cytes remain  (X  200)  (photographs  by  the  author). 

in  the  blood.  The  gametes  are  easily  recognized.  Their 
length  is  somewhat  greater  than  the  diameter  of  a  red 
corpuscle.  Their  chromatin  is  usually  centrally  placed, 
and  they  contain  more  or  less  coarse  pigment.  The  re- 
mains of  the  red  cell  often  form  a  narrow  rim  around  them 
or  fill  the  concavity  of  the  crescent. 
-  While  the  parasites  are  more  easily  found  in  stained 
preparations,  the  varieties  are  more  easily  differentiated 
in  fresh  blood.  The  chief  distinguishing  points  are 
included  in  the  table  on  page  256. 


256  THE  BLOOD 

VARIETIES   OF   THE   MALARIAL   ORGANISM 


Tertian. 

Quartan. 

EsTivo-AtrnniNAL, 

Asexual  cycle,  forty-eight 
hours. 

Seventy-two  hours. 

Usually  twenty-four  to 
forty-eight  hours. 

Substance  pale,  trans- 
parent,   comparable   to 
hyaline  tube-cast. 

Outline  indistinct. 

Ameboid  motion  ac- 
tive. 

Mature  asexual  form 
large;  fills  and  often  dis- 
tends corpuscle. 

Pigment  -granu'^s 
fine,    brown,    scattered 
throughout.      Very   ac- 
tive dancing  motion. 

Segmenting  body 
rarely   assumes   typical 
"daisy"   form.      15    to 
20  segments. 

Gametes  resemble 
asexual  forms. 

Red   corpuscles  pale 
and  swollen. 

Highly     refractive, 
comparable     to     waxy 
tube-cast. 

Distinct. 

Sluggish. 

Smaller, 

Much  coarser,  darker 
in  color,  peripherally  ar- 
ranged.   Motion  slight. 

Usually    typical 
"daisy."     6  to  12  seg- 
ments. 

Same  as  tertian. 

Generally  darker  than 
normal. 

Highly  refractive. 

Distinct. 
Active. 

Young  forms,  only, 
in  peripheral  blood. 

Very    few,    minute, 
inactive.        Distinctly 
pigmented    forms   sel- 
dom seen. 

Not    seen    in    peri- 
pheral blood. 

Appear  in  blood  as 
distinctive  ovoids  and 
crescents. 

Dark,  often  bronzed. 

4.  Filarial  Embryos.— A  description  of  the  filariae 
whose  embryos  appear  in  the  blood  will  be  found  on 
P-  356. 

The  embryos  can  be  seen  in  stained  preparations,  (Fig. 
99),  but  are  best  found  in  fresh  unstained  blood.  A 
rather  large  drop  is  taken  upon  a  slide,  covered,  and  ex- 
amined with  a  low  power.  The  embryo  can  be  located 
by  the  commotion  which   its  active  motion  produces 


SERUM  REACTIONS  257 

among  the  corpuscles.  This  motion  consists  almost 
wholly  in  apparently  purposeless  lashing  and  coiling 
movements,  and  continues  for  many  hours. 

5.  Embryos  of  Trichinella  Spiralis.— The  worm  and 
its  life-history  are  described  on  page  363.  It  has  re- 
cently been  shown  that  diagnosis  of  trichiniasis  can  fre- 
quently be  made  by  detection  of  the  embryos  in  the 
blood  during  their  migration  to  the  muscles.  Of  eleven 
such  examinations  which  have  been  reported  within  the 
past  two  and  a  half  years,  six  were  positive.  The  earliest 
time  at  which  the  embryos  were  found  was  the  sixth  day 
after  the  onset  of  symptoms;  the  latest,  the  twenty- 
second  day. 

The  method  is  very  simple.  One  to  10  c.c.  of  blood 
are  obtained  from  the  ear  or  a  vein,  as  described  on  page 
245,  and  mixed  with  ten  times  its  volume  of  3  per  cent, 
acetic  acid.  The  mixture  is  centrifugalized,  and  large 
drops  of  the  sediment  are  placed  on  sUdes,  covered,  and 
searched  with  a  low-power  objective.  The  embryos  are 
not  difficult  to  recognize.  They  are  about  1 25  |tz  long  and 
6/z  broad. 

IX.  SERUM  REACTIONS 

*I.  Agglutination.— In  the  blood-serum  of  persons 
suffering  from  certain  infectious  diseases  there  exist  sol- 
uble bodies,  called  agglutinins,  which  have  the  property  of 
rendering  non-motile  and  clumping  the  specific  micro- 
organism of  the  disease,  and  have  little  or  no  influence 
upon  other  bacteria.  This  "  agglutination  "  takes  place 
even  when  the  blood  is  greatly  diluted.  Undiluted  nor- 
mal blood  can  agglutinate  most  bacteria,  but  loses  this 
power  when  diluted  to  any  considerable  degree.    These 

17 


258 


THE   BLOOD 


facts  are  taken  advantage  of  in  the  diagnosis  of  several 
diseases. 

When  appKed  to  the  diagnosis  of  typhoid  fever,  the 
phenomenon  is  known  as  the  Widal  reaction.  As  yet,  it 
is  the  only  agglutination  reaction  which  has  any  practical 
value  for  the  practitioner. 

Either  blood-serum  or  the  whole  blood  may  be  used. 
Serum  is  the  better.  To  obtain  it,  it  is  convenient  to  use 
little  vials,  such  as  can  be  made  by  breaking  off  the  lower 


Fig.  loo. — Method  of  obtaining  blood  in  a  Wright  capsule:  A,  Filling  the  capsule;  B, 
the  bulb  has  been  warmed  and  the  capillary  end  sealed  in  a  flame;  C,  cot)ling  of  the  capsule 
has  drawn  the  blood  to  the  sealed  end;  D,  the  serum  has  separated,  and  the  top  of  the  cap- 
sule has  been  broken  off. 


half -inch  of  the  tubes  which  have  contained  pepton- 
izing powder.  They  must,  of  course,  be  well  cleaned. 
One  of  these  is  filled  to  a  depth  of  about  \  inch  from 
a  puncture  in  the  finger  or  the  ear.  and  is  set  aside  for  a 
few  hours.  When  the  clot  has  separated,  it  is  picked  out 
with  a  needle,  leaving  the  serum.  It  is,  however,  more 
satisfactory  to  obtain  the  blood  in  a  Wright  capsule 
(Fig.  loo).  This  capsule  is  easily  made  from  a  piece  of 
glass- tubing  as  indicated  in  Fig.  i6i. 


SERUM  REACTIONS  259 

One  drop  of  the  serum  is  then  added  to  nine  drops 
of  normal  salt  solution,  making  a  dilution  of  i  :  10. 
Distilled  water  may  be  used  for  dilution,  but  is  more 
liable  to  cause  error.  The  dilution  can  be  more  accu- 
rately made  in  the  leukocyte  pipet  of  the  Thoma- 
Zeiss  instrument.  When  the  whole  blood  is  used,  it 
can  be  secured  in  this  pipet  and  at  once  diluted  with 
the  salt  solution.  When  it  must  be  transported  a  con- 
siderable distance,  dried  blood  is  most  convenient.  A 
large  drop  is  allowed  to  dry  upon  a  clean  slide  or  unglazed 
paper.  It  will  keep  for  months  without  losing  its  ag- 
glutinating power.  When  ready  to  make  the  test,  the 
dried  stain  is  dissolved  in  ten  drops  of  normal  salt  solu- 
tion, care  being  taken  that  the  drops  are  about  the  same 
size  as  the  original  drop  of  blood. 

The  reaction  can  be  detected  either  microscopically  or 
macroscopically : 

Microscopic  Method. — (i)  The  blood  or  serum  having 
been  obtained  and  diluted  i  :  10  as  just  described,  mix  it  with 
a  bouillon  culture  of  the  typhoid  bacillus  to  any  desired 
dilution.  One  drop  of  each  makes  a  blood-dilution  of  i  :  20, 
etc.  The  culture  should  be  between  eighteen  and  twenty- 
four  hours  old,  and  the  bacilli  must  be  actively  motile.  A 
stock  agar  culture  should  be  kept  at  room  temperature,  and 
bouillon  tubes  inoculated  the  day  before  the  examination  is  to 
be  made.  Agar  cultures  can  be  purchased  from  dealers  in 
biologic  products.     They  must  be  renewed  monthly. 

Instead  of  the  bouillon  culture,  McFarland  recommends 
the  use  of  a  suspension  made  by  removing  some  of  the  growth 
from  the  surface  of  a  fresh  agar  culture  and  mixing  it  well 
with  a  little  sterile  water.  It  is  then  necessary  to  examine  the 
suspension  microscopically  to  make  sure  that  there  are  no 
natural  clumps. 


26o  THE    BLOOD 

(2)  Place  a  few  drops  of  the  mixture  of  blood  and  culture 
upon  a  perfectly  clean  slide  and  apply  a  cover-glass.  The 
cover  may  be  ringed  with  vaselin  to  prevent  evaporation,  but 
this  is  not  usually  necessary. 

(3)  Examine  at  intervals  with  a  high  dry  lens — a  4  mm. 
will  answer  very  well.  The  light  must  be  very  subdued. 
At  first  the  bacilli  should  be  actively  moving  about.  If  the 
blood  be  from  a  case  of  typhoid,  they  will  gradually  lose  their 
motion  and  gather  together  in  clumps  (Fig.  loi).  The  clumps 
should  be  large,  and  the  few  bacilli  remaining  isolated  should 


Fig.  101. — Showing  clumping  of  typhoid  bacilli  in  the  Widal  reaction.    At  one  point  a 
crenated  red  blood-corpuscle  is  seen  (Wright  and  Brown). 

be  motionless.  Pseudoreactions,  in  which  there  are  a  few 
small  clumps  of  bacilli  whose  motion  is  not  entirely  lost, 
together  with  many  freely  moving  bacilli  scattered  through- 
out the  field,  should  not  mislead.  As  a  control,  a  drop 
of  the  culture  should  always  be  examined  before  making 
the  test. 

Normal  blood  may  produce  clumping  if  time  enough  be 
allowed.  The  diagnostic  value  of  a  positive  reaction  is,  there- 
fore, impaired  unless  clumping  takes  place  within  a  limited 
time.     With  dilution  of  i  :  40  the  time  limit  should  not  exceed 


SERUM  REACTIONS  26 1 

forty-five  minutes;  with  i  :  80,  one  and  one-half  hours. 
Tests  based  upon  lower  dilution  than  i  :  40  are  probably  not 
reliable. 

Macroscopic  Method. — The  principle  is  the  same  as  that 
of  the  microscopic  method.  Clumping  of  the  bacilli  causes 
a  flocculent  precipitate,  which  can  be  seen  with  the  naked 
eye.  A  dead  culture  gives  the  same  results  as  a  living  one. 
This  method  is  as  reliable  as  the  microscopic  and  is  more 
convenient  for  the  practitioner,  although  it  requires  more 
time. 

Dead  cultures,  together  with  apparatus  for  diluting  the 
blood,  are  put  up  at  slight  cost  by  various  firms,  under  the 
names  of  typhoid  diagnosticum,  typhoid  agglutometer,  etc. 
Full  directions  accompany  these  outfits  and  need  not  be 
repeated  here. 

Recently,  Bass  and  Watkins  have  described  a  modification 
of  the  macroscopic  method  (using  very  concentrated  sus- 
pensions of  the  bacilli)  by  which  the  test  can  be  applied  at 
the  bedside.  Clumping  occurs  within  two  minutes, '  The 
apparatus  has  been  put  upon  the  market  by  Parke, 
Davis  &  Co. 

The  Widal  reaction  is  positive  in  over  95  per  cent,  of  all 
cases  of  typhoid  fever.  It  may,  rarely,  be  positive  in 
other  conditions,  owing,  sometimes  at  least,  to  faulty 
technic.  It  seldom  appears  before  the  fifth  or  sixth  day; 
usually  during  the  second  week,  but  sometimes  not  until 
convalescence.  It  is,  therefore,  of  less  value  in  early 
diagnosis  than  is  the  blood-culture  (p.  244).  When  it 
once  appears  it  remains  during  the  whole  course  of  the 
disease,  and  frequently  persists  for  years. 

2.  Opsonins. — That  phagocytosis  plays  an  important 
part  in  the  body's  resistance  to  bacterial  invasion  has 
long  been  recognized.    According  to  Metchnikoff,  this 


262  THE  BLOOD 

property  of  leukocytes  resides  entirely  within  themselves, 
depending  upon  their  own  vital  activity.  The  studies 
of  Wright  and  Douglas,  upon  the  contrary,  indicate 
that  the  leukocytes  are  impotent  in  themselves,  and  can 
ingest  bacteria  only  in  the  presence  of  certain  substances 
which  exist  in  the  blood-plasma.  These  substances  have 
been  named  opsonins.  Their  nature  is  undetermined. 
They  probably  act  by  uniting  with  the  bacteria,  thus 
preparing  them  for  ingestion  by  the  leukocytes ;  but  they 
do  not  cause  death  of  the  bacteria,  nor  produce  any 
appreciable  morphologic  change.  They  appear  to  be 
more  or  less  specific,  a  separate  opsonin  being  necessary 
for  phagocytosis  of  each  species  of  bacteria.  There  are, 
moreover,  opsonins  for  other  formed  elements — red 
blood-corpuscles,  for  example.  It  has  been  shown  that 
the  quantity  of  opsonins  in  the  blood  can  be  greatly 
increased  by  inoculation  with  dead  bacteria. 

To  measure  the  amount  of  any  particular  opsonin  in  the 
blood  Wright  has  devised  a  method  which  involves  many 
ingenious  and  delicate  technical  procedures.  Much  skill, 
such  as  is  attained  only  after  considerable  training  in  lab- 
oratory technic,  is  requisite,  and  there  are  many  sources 
of  error.  It  is,  therefore,  beyond  the  province  of  this 
work  to  recount  the  method  in  detail.  In  a  general  way 
it  consists  in:  {a)  Preparing  a  mixture  of  equal  parts  of 
the  patient's  blood-serum,  an  emulsion  of  the  specific 
micro-organism,  and  a  suspension  of  washed  leukocytes; 
{h)  preparing  a  similar  mixture,  using  serum  of  a  normal 
person;  {c)  incubating  both  mixtures  for  a  definite  length 
of  time;  and  id)  making  smears  from  each,  staining,  and 
examining  with  an  oil-immersion  objective.  The  num- 
ber of  bacteria  which  have  been  taken  up  by  a  definite 


SERUM  REACTIONS  263 

number  of  leukocytes  is  counted,  and  the  average  number 
of  bacteria  per  leukocyte  is  calculated;  this  gives  the 
"phagocytic  index."  The  phagocytic  index  of  the  blood 
under  investigation,  divided  by  that  of  the  normal 
blood,  gives  the  opsonic  index  of  the  former,  the  opsonic 
index  of  the  normal  blood  being  taken  as  i.  Simon  re- 
gards the  percentage  of  leukocytes  which  have  ingested 
bacteria  as  a  more  accurate  measurement  of  the  amount 
of  opsonins  than  the  number  of  bacteria  ingested,  be- 
cause the  bacteria  are  apt  to  adhere  and  be  taken  in  in 
clumps. 

Because  of  its  simplicity  the  clinical  laboratory 
worker  will  prefer  some  modification  of  the  Leishman 
method,  which  uses  the  patient's  own  leukocytes.  It 
is,  perhaps,  as  accurate  as  the  original  method  of  Wright, 
although  variations  in  the  leukocyte  count  have  been 
shown  to  affect  the  result.  Two  pipets  like  those 
shown  in  Fig.  164  are  used. 

(i)  Make  a  suspension  of  the  specific  organism  by  mixing 
a  loopful  of  a  young  agar  culture  with  i  c.c.  of  a  solution  con- 
taining I  per  cent,  sodium  citrate  and  0.85  per  cent,  sodium 
chlorid.  Thoroughly  break  up  all  clumps  by  sucking  the 
fluid  in  and  forcing  it  out  of  one  of  the  capillary  pipets  held 
vertically  against  the  bottom  of  the  watch-glass. 

(2)  Puncture  the  patient's  ear,  wipe  off  the  first  drop  of 
blood,  and  from  the  second  draw  blood  into  the  other  pipet 
to  the  grease  pencil  mark,  let  in  a  bubble  of  air,  and  draw  in 
-the  same  amount  of  bacterial  suspension. 

(3)  Mix  upon  a  slide  by  drawing  in  and  forcing  out  of  the 
pipet. 

(4)  Draw  the  mixture  high  up  in  the  pipet,  seal  the  tip 
in  the  flame,  and  place  in  the  incubator  for  fifteen  minutes. 


264  THE   BLOOD 

(5)  Repeat  steps  2,  3,  and  4  with  the  blood  of  a  normal 
person. 

(6)  After  incubation,  break  off  the  tip  of  the  pipet,  mix  the 
blood-bacteria  mixture,  and  spread  films  on  slides. 

(7)  Stain  with  Wright's  or  Harlow's  blood-stain. 

(8)  With  an  oil-immersion  lens  count  the  bacteria  which 
have  been  taken  in  by  100  leukocytes,  and  calculate  the  aver- 
age number  per  leukocyte.  Divide  the  average  for  the 
patient  by  the  average  for  the  normal  person.  This  gives 
the  opsonic  index.  If  in  the  patient's  blood  there  was  an 
average  of  4  bacteria  per  leukocyte,  and  in  the  normal  blood 
5  bacteria  per  leukocyte,  the  opsonic  index  would  be  |  or  0.8. 

Wright  and  his  followers  regarded  the  opsonic  index 
as  an  index  of  the  power  of  the  body  to  combat  bacterial 
invasion.  They  claimed  very  great  practical  importance 
for  it  as  an  aid  to  diagnosis  and  as  a  guide  to  treatment 
by  the  vaccine  method.  This  method  of  treatment  con- 
sists in  increasing  the  amount  of  protective  substances 
in  the  blood  by  injections  of  normal  salt  suspensions  of 
dead  bacteria  of  the  same  species  as  that  which  has 
caused  and  is  maintaining  the  morbid  process,  these 
bacterial  suspensions  being  called  'Vaccines."  Vaccine 
Therapy  (Chapter  IX)  has  taken  a  permanent  place 
among  our  methods  of  treatment  of  bacterial  infections, 
particularly  of  those  which  are  strictly  local,  but  the 
opsonic  index  is  now  little  used  either  as  a  measure  of 
resisting  power  or  as  an  aid  to  diagnosis  and  guide  to 
treatment. 

3.  Wassermann  Reaction.'— The  Wassermann  test 
for  syphilis,  like  the  Widal  test  for  typhoid  fever,  de- 

*  By  Clough  T.  Burnett,  Professor  of  Bacteriology,  University  of  Col- 
oradg. 


SERUM  REACTIONS  265 

pends  upon  the  detection  in  the  patient's  blood-serum  of 
specific  antibodies,  agglutinins  in  the  case  of  typhoid, 
immune  bodies  or  amboceptors  in  the  case  of  syphilis. 
These  antibodies  have  been  produced  by  the  tissues  in 
response  to  the  entrance  of  the  invading  organism.  If 
they  are  present,  it  is  assumed  that  the  patient  has  or 
has  had  syphiHs.  The  Wassermann  test  is,  however, 
much  more  complicated  than  the  Widal  test,  and  can  be 
properly  performed  only  by  a  trained  laboratory  worker. 
It  is  the  aim  here  to  explain  only  the  general  principles 
of  the  method,  together  with  its  clinical  significance. 
For  a  proper  understanding  of  the  test  the  principles 
of  bacteriolysis  and  hemolysis  must  first  be  presented. 

Bacteriolysis  and  Hemolysis. — In  1894  Pfeiffer,  work- 
ing with  guinea-pigs  immunized  to  cholera,  found  that 
when  living  cholera  germs  were  introduced  into  the 
peritoneal  cavity  of  an  immune  animal  they  lost  their 
motility  within  a  few  minutes,  and  very  shortly  were 
seen  to  disintegrate  and  go  into  complete  solution. 
This  has  been  known  as  Pfeiffer's  phenomenon,  or 
bacteriolysis.  It  was  later  demonstrated  that  this  reac- 
tion could  take  place  outside  the  animal  body  if  the 
bacteria  were  mixed  in  the  test-tube  with  the  blood- 
serum  or  peritoneal  fluid  of  a  cholera  immune  animal. 
Subsequent  researches  showed  that  while  an  old  or 
heated  immune  serum  failed  to  cause  this  solution  of 
the  bacteria,  upon  the  addition  of  a  normal  fresh  serum 
this  property  returned.  This  addition  of  a  normal 
serum  to  a  serum  which  has  lost  its  solvent  action  is 
called  reactivation  of  the  serum.  These  changes  may 
best  be  demonstrated  by  the  following  chart; 


266 


3 

THE   BLOOD 

Bacteriolysis. 

Immune  serum, 

fresh 

+  bacteria            =  solution. 

Normal       " 

" 

+        "                 =  no  solution. 

Immune      " 

heated 

+        "                  =  no  solution. 

Immune      " 

" 

+  normal  serum  +  bacteria  =  solution. 

From  the  chart  it  is  clear  that  there  are  two  sub- 
stances concerned  in  bacteriolysis,  one  of  which  is  found 
in  any  fresh  serum,  but  is  easily  destroyed,  and  is  called 
the  complement.  The  other  substance  is  found  only  in 
the  immune  serum,  is  relatively  stable,  and  is  known  as 
the  immune  body  or  amboceptor. 

In  hemolysis  we  find  an  analogy  to  bacteriolysis. 
Let  a  rabbit  be  immunized  to  sheep's  blood-corpuscles. 
Now,  if  washed  sheep's  blood-corpuscles  be  subjected  to 
the  action  of  fresh  serum  from  this  rabbit,  a  speedy  so- 
lution of  the  red  cells  ensues.  If  this  serum  is  allowed 
to  stand  for  several  days,  or  is  heated  one-half  hour  to 
56°  C,  it  will  completely  lose  its  solvent  power.  Now, 
the  addition  of  a  fresh  normal  serum,  even  of  another 
species,  will  reactivate  the  heated  or  old  serum.  The 
following  chart  will  indicate  these  reactions: 

Hemolysis. 
Rabbit  serum,  immune  -f-  corpuscles  (sheep's)  =  solution. 

Rabbit      "  "  heated  +  "  "         =  no  solution. 

Normal      "  +  "  ''         =  no  solution. 

Rabbit      "  "        heated  +  normal  serum  +  corpuscles  (sheep's) 

=  solution. 

In  hemolysis,  as  in  bacteriolysis,  besides  the  antigen 
(substance  giving  rise  to  amboceptors  or  antibodies)  there 
are  two  substances.  One  of  these  is  specific,  /.  e.,  only 
found  in  immune  serum,  and  reacting  only  with  the  sub- 
stance used  in  producing  the  immune  serum.     This  sub- 


SERUM  REACTIONS  267 

stance  is  relatively  stable,  and  is  known  as  the  amboceptor. 
The  other  substance  is  found  in  any  serum,  is  absolutely 
non-specific,  is  easily  destroyed,  and  is  called  the  com- 
plement. In  neither  case  will  the  amboceptor  nor  the 
complement  acting  alone  cause  a  solution  of  the  antigen. 

There  are  three  substances  necessary  to  bacteriolysis 
and  hemolysis.  To  produce  bacteriolysis  there  must  be 
the  specific  antigen  (as  cholera  vibrio  in  Pfeififer's  phe- 
nomenon), the  immune  serum  containing  the  amboceptor, 
and  a  complement.  These  three  substances  comprise 
the  bacteriolytic  system.  Likewise,  in  hemolysis  there 
is  the  red  blood-cell,  the  amboceptor,  and  a  complement, 
which  comprise  the  hemolytic  system. 

It  will  be  noted  that  there  is  one  factor  common  to 
both  systems,  viz.,  the  complement.  It  will  be  evident 
that  if  we  place  in  a  test-tube  a  complete  bacteriolytic 
system,  with  just  enough  complement  to  cause  solution 
of  the  bacteria,  and  place  this  for  a  sufficient  time  in 
the  optimum  temperature  for  bacteriolysis,  and  then  add 
two  elements  of  the  hemolytic  system  (amboceptor  and 
blood-cells),  no  hemolysis  will  ensue,  because  all  of  the 
complement  was  used  by  the  bacteriolytic  system. 

Bordet  and  Gengou  in  1901  showed  that  it  was  pos- 
sible to  utilize  this  fact  in  the  diagnosis  of  certain  bac- 
terial infections.  For  instance,  the  heated  serum  of  a 
suspected  typhoid  case  plus  typhoid  bacilli  is  added 
to  a  serum  containing  complement  (fresh  guinea-pig 
serum)  and  incubated  one  hour.  If  this  suspected  serum 
contains  typhoid  amboceptors,  there  will  have  been 
a  combination  effected  between  the  three  elements  of 
the  bacteriolytic  system,  so  that  there  will  be  no  free 
complement  left.     If  no  amboceptor  is  present,  all  of 


268 


THE   BLOOD 


the  complement  will  remain  unattached.  Now,  in 
order  to  show  whether  this  complement  has  been  fixed 
or  deviated,  two  elements  of  a  hemolytic  system  are 
added — namely,  amboceptor  and  red  corpuscles,  and  if 
the  complement  is  fixed,  no  hemolysis  can  ensue. 

This  is  easily  understood  from  a  study  of  the  swinging 
pendulum  diagram,  in  which  the  complement  is  repre- 
sented in  the  pendulum. 


DACTERIOLYTIC 
SYSTEM 


HEMOL>/TI.C 
SYSTEM 


Fig.  I02. — Pendulum  diagram  illustrating  hemolysis.' 

This  principle  of  "complement  deviation"  having  been 
utilized  in  the  diagnosis  of  infectious  diseases,  it  occurred 
to  Wassermann  in  1906  to  apply  it  to  the  diagnosis  of 
syphilis.  The  antigen  first  used  was  the  extract  of  a  fetal 
syphilitic  liver,  but  subsequent  work  has  shown  that  the 
same  reaction  may  be  obtained  with  normal  liver  or 
spleen  tissue,  or  with  certain  lipoid  substances,  and  in 
this  sense  is  not  a  true  antigen-antibody  reaction.  The 
antibodies  in  the  syphilitic  blood,  however,  are  specific. 
These  are  analogous  to  the  bacteriolytic  amboceptor  of 
the  pendulum  diagram. 

*  Not  original,  but  unable  to  place  credit  where  due. 


SERUM  REACTIONS  269 

Technic  of  the  Wassennann  Test. — ^The  following  reagents 
are  necessary: 

Antigen. — Extract  of  fetal  syphilitic  or  normal  liver, 
diluted  I  :  10. 

Antibodies  (Analogous  to  Bacteriolytic  Antibodies). — The 
serum  or  spinal  fluid  of  the  suspected  patient.  As  controls, 
the  serum  of  a  syphilitic  known  to  contain  antibodies  and  the 
serum  of  a  normal  person  known  not  to  contain  antibodies. 

Complement. — Fresh  guinea-pig  serum.  Other  fresh  nor- 
mal sera  may  be  used.    This  is  diluted  i  :  10. 

Hemolytic  Amboceptor. — The  serum  of  a  rabbit  which  has 
been  immunized  to  sheep's  red  blood-corpuscles.  This  serum 
is  inactivated  before  use  by  heating  to  56°  C.  one-half  hour, 
and  diluted  i  :  1000  before  using. 

Corpuscle  Suspension. — Sheep's  blood  is  defibrinated, 
washed  three  times  with  normal  salt  solution,  and  then 
diluted  with  normal  salt  solution  to  make  a  5  per  cent,  sus- 
pension. 

In  using  these  various  reagents  it  is  necessary  to  know  that 
they  are  potent  and  of  the  proper  strength,  that  is,  to  establish 
the  titre  of  the  reagent.  This  being  determined,  we  are  now 
ready  for  the  Wassermann  test,  as  carried  out  in  the  table 
on  page  270. 

In  the  luetic  control  tube  there  will  occur  a  combination 
between  the  antibodies  in  the  serum  and  the  antigen,  which 
together  will  cause  a  fixation  of  the  complement,  so  that 
when  later  the  two  elements  of  the  hemolytic  system  are 
added,  no  hemolysis  will  occur.  This  inhibition  of  hemolysis 
indicates  a  positive  syphilitic  reaction. 

Control  tube  No.  3  is  used  to  show  that  there  is  nothing 
in  a  normal  serum  which  can  effect  this  combination  and 
deviation.  Tube  No.  4  shows  that  the  patient's  serum  alone 
is  not  anticomplementary.  Tube  No.  5  shows  that  the 
hemolytic  system  is  effective.    Tube  No.  6  shows  that  the 


270 


THE  BLOOD 


h) 

"' 

0 

u 

(- 

z 

a 

0 

H 

u 

►j 

0 

z 

0 

H 

u 

o  ^j 


d  d  <N 


E    -^5 

—    y    O 

o  eg 


g2g- 


CO 

<N     ■*   '^   Tj- 

d  d  d  "N 


-^  E 


E    .-3 

-SCO 
6    d    r^t 


E 


I   -t  "t  o 

1  d  d  rl 


03    ""    O    C-^ 


°.  ° 

M     M 

C 

i^'"-2 

«  *j 

■^-2 

3  S 

e-- 

^'3^ 

M     0  00 

d  "  d 

u        c 

2  «.2 

ocep 
uscle 
solut 

<CJcAi 

<M     0   00 

6  >^  6 

C      c 

ocepto 
uscles, 
solutio 

u 

S  0  ^ 

0 

<uJ5 

"0 

<N     0  00 

rt 

d  w  d 

3 
0 

0 
C 

oceptor, 

uscles, 

solution, 

0 

E  o-:^ 

.£2 

3 

<  CJ  v: 

<N     0  00 

0 
C 
1— 1 

d  "  d 

C      c 

s^  ■?;  — 

u  2  0 

0   3   5] 

E  013 

<Ut^ 

ts    000 

d  M  d 

uT      a 

ocepto 
uscles, 
solutio 

E  0 -^ 

<uc5? 

IN     0  00 

d  "  d 

«         ^ 

tT-     C 

0   ,^'  0 

ocept 
uscle; 
soluti 

6  S^ 

<Uc« 

0 

x> 

ti 

S 

u 

eS 

Xi 

V 

0 

j: 

^ 

li 

o      i_        j:? 


o    2 


""I       -^1 


t>         .i' 


:S  :_ffi_ 


-^      .23 


="  ^  Si 

a  2  a 

I  u» 

53  -00 

U  U    '^ 

.£  -S  <« 

S  1:; 

0  .  C  4> 
X3  O  «  J3 
3  "   S^   3 

^  M     «     S 

•  •  -  o  5 

.2  c  *J  SJ 

1  c2  ■" 

0) 


o  — 


o  c 


?   u 


(U.2    U    u^ 

J=  ^  J= 
HHH< 


SERUM  REACTIONS  27 1 

antigen  alone  is  not  anticomplementary.  Tube  No,  7  is 
introduced  to  show  that  hemolysis  will  not  occur  in  the  ab- 
sence of  the  complement. 

Modifications. — Certain  modifications  of  this  test 
have  been  suggested,  chief  of  which  is  the  Noguchi  test. 
This  differs  from  the  Wassermann  mainly  in  that  an  anti- 
human  hemolytic  system  is  used  instead  of  an  anti- 
sheep,  because,  according  to  the  author,  there  is  an 
appreciable  error  in  the  Wassermann  in  that  there  is 
present  "  in  human  serum  a  variable  amount  of  natural 
antisheep  amboceptor "  capable  of  so  changing  the 
results  that  with  sera  containing  only  a  small  amount 
of  syphilitic  antibody  the  result  will  be  negative. 

The  following  reagents  are  used : 

1.  Antihuman  hemolytic  amboceptor  prepared  by  re- 
peated injections  of  a  rabbit  with  washed  human  blood- 
corpuscles. 

2.  Complement.     Fresh  guinea-pig  serum. 

3.  Antigen.    Organ  extracts  or  a  solution  of  lecithin. 

4.  I  per  cent,  suspension  of  human  blood-corpuscles. 

5.  The  suspected  serum. 

6.  A  known  syphilitic  serum. 

7.  A  serum  known  not  to  contain  syphilitic  antibodies. 
With  these  reagents  the  procedure  is  very  little  different 

from  that  outlined  for  the  Wassermann  test. 

Noguchi  has  further  simplified  it  for  the  small  laboratory 
by  drying  the  amboceptor  serum  on  slips  of  filter-paper, 
which  can  be  kept  a  considerable  time.  The  same  procedure 
can  be  carried  out  with  the  antigen.  While  at  first  similar 
complement  slips  were  prepared,  it  is  now  known  that  fresh 
complement  is  indispensable. 


272 


THE   BLOOD 


Value  of  Wassermann  Test. — The  reaction  is  positive 
in  95  to  98  per  cent,  of  all  cases  with  syphilitic  manifes- 
tations. In  the  late  cases  only  a  very  slight  inhibition 
of  hemolysis  may  be  noted.  This  has  given  rise  to 
considerable  difficulty  in  the  interpretation  of  results. 
Kaplan  states  that  the  report  should  read  "negative" 
or  "  positive,"  with  no  report  of  the  degree  of  inhibition. 

Butler  obtains  the  following  results: 

No.  of  Per  cent. 

cases.  positive. 

Controls,  non-syphilitic 53  o 

Primary  syphilitic 10  100 

Secondary  syphilitic 36  95 

Tertiary  syphiltic 31  94 

Latent  cases 16  56 

Parasyphilis  and  visceral  syphilis 55  76 

Total  cases 201 

Kaplan,  in  a  study  of  diseases  of  the  nervous  system, 
obtained  the  following  results:  In  249  cases  of  quiescent 
tabes  the  Wassermann  reaction  was  positive  in  44  per 
cent.;  in  57  cases  of  active  tabes,  88  per  cent.,  and  in  64 
cases  of  general  paresis,  88  per  cent. 

By  the  Noguchi  method  about  7  per  cent,  of  non- 
syphilitic  sera  will  cause  inhibition  of  hemolysis,  while 
with  the  Wassermann,  in  about  9  per  cent,  of  known 
syphilitic  sera,  hemolysis  will  occur.  For  this  reason  in 
doubtful  cases  it  is  well  to  apply  both  methods. 

It  is  probable  that  a  positive  reaction  almost  always 
means  active  syphilis  even  without  manifestations,  but 
it  is  not  absolutely  specific  for  syphilis,  for  the  reaction 
has  been  obtained  in  leprosy.  Kaplan  states  that  "  old 
leprosy  cases  present  a  much  more  definitely  positive 
reaction  than  cases  of  old  syphiHs."  Many  workers 
believe  that  a  positive  reaction  in  a  late  case  may  only 


SERUM  REACTIONS  273 

indicate  that  the  patient  has  once  had  syphilis.  Against 
this  view  stands  the  fact  that  in  other  infectious  diseases 
antibodies  diminish  or  entirely  disappear  a  few  months 
after  the  active  infection,  and  that  in  latent  cases  the 
reaction  may  disappear  under  treatment.  On  the  other 
hand,  there  are  certain  cases  which  are  considered  clinic- 
ally as  cured,  and  have  remained  so  for  years,  that  will 
continue  to  give  the  positive  reaction  in  spite  of  any 
treatment. 

In  the  application  of  the  test  to  the  diagnosis  of  dis- 
eases of  the  nervous  system  and  viscera  one  should  al- 
ways bear  in  mind  the  possibility  of  a  dual  pathologic 
process,  and  a  positive  test  should  not  be  allowed  to 
entirely  overshadow  the  clinical  findings. 

Effect  of  Treatment. — The  positive  reaction  fre- 
quently disappears  after  a  short  course  of  treatment 
with  mercury.  This  may  be  permanent,  or,  after  a 
variable  length  of  time,  the  reaction  may  return.  Some 
cases  thoroughly  treated  persist  in  giving  a  positive 
reaction.  In  hereditary  syphilis  it  is  often  impossible 
to  get  rid  of  the  reaction.  Because  the  reaction  may 
return,  it  is  always  safer  to  make  several  tests  before 
deciding  that  further  treatment  is  not  indicated.  This 
suggests  that  while  a  positive  reaction  may  be  accepted 
as  an  indication  of  syphilis,  a  negative  reaction  obtained 
after  treatment  may  not  exclude  syphilis. 

Following  treatment  with  salvarsan  {"  606  "),  Noguchi, 
in  a  total  of  102  cases,  finds  that  the  positive  reaction 
becomes  negative  in  33.7  per  cent. 

18 


274  THE   BLOOD 

X.  TESTS  FOR  RECOGNITION  OF  BLOOD 

1.  Quaiac  Test.— The  technic  of  this  test  has  been 
given  (p.  125).  It  may  be  appHed  directly  to  a  suspected 
fluid  or,  better,  to  the  ethereal  extract.  Add  a  few 
cubic  centimeters  of  glacial  acetic  acid  to  about  10  c.c. 
of  the  fluid;  shake  thoroughly  with  an  equal  volume  of 
ether;  decant,  and  apply  the  test  to  the  ether.  In  case 
of  dried  stains  upon  cloth,  wood,  etc.,  dissolve  the  stain 
in  distilled  water  and  test  the  water,  or  press  a  piece  of 
moist  blotting-paper  against  the  stain,  and  touch  the 
paper  with  drops  of  the  guaiac  and  the  turpentine  suc- 
cessively. 

2.  Teichmann's  Test.— This  depends  upon  the  pro- 
duction of  characteristic  crystals  of  hemin.    It  is  a  sensi- 


Fig.  103. — Teichmann's  hemin  crystals  (Jakob). 

tive  test  and,  when  positive,  is  absolute  proof  of  the 
presence  of  blood.  A  number  of  substances — lime,  fine 
sand,  iron  rust — interfere  with  production  of  the  crys- 
tals; hence  negative  results  are  not  always  conclusive. 
Dissolve  the  suspected  stain  in  a  few  drops  of  normal 


SPECIAL  BLOOD  PATHOLOGY  275 

salt  solution  upon  a  slide.  If  a  liquid  is  to  be  tested, 
evaporate  some  of  it  upon  a  slide  and  dissolve  the  residue 
in  a  few  drops  of  the  salt  solution.  Let_dry,  apply  a 
cover-glass,  and  run  glacial  acetic  acid  underneath  it. 
Heat  mry  gently  until  "bubbles  begin  to  form,  replacing 
the  acid  as  it  evaporates.  Allow  to  cool  slowly.  When 
cool,  replace  the  acid  with  water,  and  examine  for 
hemin  crystals  with  16  mm.  and  4  mm.  objectives.  The 
crystals  are  dark-brown  rhombic  plates,  lying  singly 
or  in  crosses,  and  easily  recognized  (Fig.  103).  Failure 
to  obtain  them  may  be  due  to  too  great  heat  or  too  rapid 
cooling.  If  not  obtained  at  first  let  the  slide  stand  in  a 
warm  place,  as  upon  a  hot-water  radiator,  for  an  hour. 

XI.  SPECIAL  BLOOD  PATHOLOGY 

The  more  conspicuous  characteristics  of  the  blood  in 
various  diseases  have  been  mentioned  in  previous  sec- 
tions. Although  the  great  majority  of  blood  changes  are 
secondary,  there  are  a  few  blood  conditions  in  which  the 
changes  are  so  prominent,  or  the  etiology  so  obscure,  that 
they  are  commonly  regarded  as  blood  diseases.  These 
will  receive  brief  consideration  here. 

A.     Anemia 

This  is  a  deficiency  of  hemoglobin,  or  red  corpuscles, 
or  both.  It  is  either  primary  or  secondary.  The  dis- 
tinction is  based  chiefly  upon  etiology,  although  each 
'type  presents  a  more  or  less  distinctive  blood-picture. 
Secondary  anemia  is  that  which  is  symptomatic  of  some 
other  pathologic  condition.  Primary  anemia  is  that 
which  progresses  without  apparent  cause. 


276  THE   BLOOD 

1.  Secondary  Anemia.— The  more  important  condi- 
tions which  produce  secondary  or  symptomatic  anemia 
are: 

(a)  Poor  nutrition,  which  usually  accompanies  unsani- 
tary conditions,  poor  and  insufficient  food,  etc. 

{b)  Acute  infectious  diseases,  especially  rheumatism 
and  typhoid  fever.  The  anemia  is  more  conspicuous 
during  convalescence. 

(c)  Chronic  Infectious  Diseases. — Tuberculosis,  mala- 
ria, syphilis,  leprosy. 

{d)  Chronic  exhausting  diseases,  as  heart  disease, 
chronic  nephritis,  cirrhosis  of  the  liver,  and  gastro- 
intestinal diseases,  especially  when  associated  with 
atrophy  of  gastric  and  duodenal  glands.  The  last  may 
give  an  extreme  anemia,  indistinguishable  from  perni- 
cious anemia. 

{e)  Chronic  poisoning,  as  from  lead,  arsenic,  and 
phosphorus. 

(/)  Hemorrhage. — Either  repeated  small  hemorrhages, 
as  from  gastric  cancer  and  ulcer,  uterine  fibroids,  etc., 
or  a  single  large  one. 

{g)  Malignant  Tumors. — These  affect  the  blood  partly 
through  repeated  small  hemorrhages,  partly  through 
toxic  products,  and  partly  through  interference  with 
nutrition. 

{h)  Animal  Parasites. — Some  cause  no  appreciable 
change  in  the  blood;  others,  like  the  hookworm  and 
Dihothriocephalus  latus,  may  produce  a  very  severe 
anemia,  almost  identical  with  pernicious  anemia.  Ane- 
mia in  these  cases  is  probably  due  both  to  toxins  and  to 
abstraction  of  blood. 

The  blood-picture  varies  with  the  grade  of  anemia. 


SPECIAL   BLOOD  PATHOLOGY  277 

Diminution  of  hemoglobin  is  the  most  characteristic 
feature.  In  mild  cases  it  is  slight,  and  is  the  only  blood 
change  to  be  noted.  In  very  severe  cases  hemoglobin 
may  fall  to  15  per  cent.  Red  corpuscles  are  diminished 
in  all  but  very  mild  cases,  while  in  the  severest  cases 
the  red  corpuscle  count  is  sometimes  below  2,000,000. 
The  color-index  is  usually  decreased. 

Although  the  number  of  leukocytes  bears  no  relation 
to  the  anemia,  leukocytosis  is  common,  being  due  to  the 
same  cause. 

Stained  films  show  no  changes  in  very  mild  cases.  In 
moderate  cases  variations  in  size  and  shape  of  the  red 
cells  and  polychromatophilia  occur.  Very  severe  cases 
show  the  same  changes  to  greater  degree,  with  addition 
of  basophilic  degeneration  and  the  presence  of  normo- 
blasts in  small  or  moderate  numbers.  Megaloblasts 
in  very  small  numbers  have  been  encountered  in  ex- 
tremely severe  cases.  They  are  especially  abundant 
and  may  even  predominate  over  the  normoblasts  in 
dibothriocephalus  infection.  Blood-plaques  are  usually 
increased. 

2.  Primary  Anemia. — The  commonly  described  vari- 
eties of  primary  anemia  are  pernicious  anemia  and  chlo- 
rosis, but  splenic  anemia  may  also  be  mentioned  under 
this  head. 

(i)  Progressive  Pernicious  Anemia. — It  is  frequently 
impossible  to  diagnose  this  disease  from  the  blood  ex- 
amination alone.  Severe  secondary  anemia  sometimes 
gives  an  identical  picture.  Remissions,  in  which  the 
blood  approaches  the  normal,  are  common.  All  the 
clinical  data  must,  therefore,  be  considered. 

Hemoglobin  and  red  corpuscles  are  always  greatly 


278 


THE   BLOOD 


diminished.  In  none  of  Cabot's  139  cases  did  the  count 
exceed  2,500,000,  the  average  being  about  1,200,000. 
In  more  than  two-thirds  of  the  cases  hemoglobin  was 
reduced  to  less  extent  than  the  red  corpuscles;  the  color- 
index  was,  therefore,  high.  A  low  color-index  probably 
indicates  a  mild  type  of  the  disease. 


Fig.  104. — A,  Normal  blood;  B,  chlorosis;  C,  pernicious  anemia.  The  plate  shows 
the  sharp  contrast  between  cells  rich  in  hemoglobin  and  the  pale  cells  of  chlorosis,  and  also 
the  poikilocytes  and  marked  variations  in  size  noted  in  pernicious  anemia.  A  normo- 
blast and  megaloblast  also  appear.     Stained  smears  (from  Greene's  "Medical  Diagnosis"). 

The  leukocyte  count  may  be  normal,  but  is  commonly 
diminished  to  about  3000.  The  decrease  affects  chiefly 
the  polymorphonuclear  cells,  so  that  the  lymphocytes  are 
relatively  increased.  In  some  cases  a  decided  absolute 
increase  of  lymphocytes  occurs.  Polymorphonuclear 
leukocytosis,  when  present,  is  due  to  some  complication. 


■  iHtfa-UM  A  ,*al^:>oi£j^tn  ,i  .iiuiji-i-^aj^    ^ 


fwi    l«nnon 


/:)jv  nnilj/  I 


-9)fus  8VBb  bv/J  obiun  noiJciaqsiq  rfaoloija  nwon^Jnu  'to  Bimsnc  guobrn 
-orrnori"?,^  b3siTD):)t;iBfI')  89haoqioo-ix)o[d   bai   b3JB3hu7:   ,u     trriariorn 

(ijiiii  ioviEiJ  .''b  bnK  'u  ;iohun  oril  \o  gnixiUje  -^en-jJni  orfj  -{d  eIebW 
,.(ij  "i     A     •siisjDriuq  vbnft   m2£[qo)(nq   'nii   ;a-jJ'poirf)vT)   ni  Z'jiuafl' 

lo  '181BOJ  H)iv>  2}gBMoiritvi9  ,-i  ;t?.£JdolBg9rn  n  ni  eiavlo-^iB;! 

■  nfiiTj    vnH    lint    f^"!    ?.nfr,rTT?    ir.'-'h:jr.    ,V>    ;rr»R!qo)mq   9rf} 


(^in«si!vl-l9ac(i 


Explanation  of  Plate  IX 

Fig.  I. — Preparation  from  an  advanced  case  of  progressive  perni- 
cious anemia  from  unknown  cause:  a,  Megaloblasts  or  gigantoblasts;  the 
protoplasm  shows  marked  polychromasia;  b,  stained  granules  in  erjlhro- 
cytes  with  normally  stained  protoplasm;  c  and  d,  polychromatophilic 
degeneration;  e,  megalocytes;  /,  normocytes. 

Fig.  2. — Preparation  from  the  same  case  taken  somje  time  later 
whUe  the  patient  was  subjectively  and  objectively  in  perfect  health: 
ii,  Punctate  erythrocytes  with  normal  and  anemic  degenerated  proto- 
plasm; b,  polynuclear  leukocj'te;  c,  normal  red  blood-corpuscles;  d, 
somewhat  enlarged  erythrocytes. 

Fig.  3. — Series  of  cells  from  a  case  of  severe  progressive  per- 
nicious anemia  of  unknown  etiology;  preparation  made  two  days  ante- 
mortem:  a.  Nucleated  red  blood-corpuscles  characterized  as  normo- 
blasts by  the  intense  staining  of  the  nuclei;  a'  and  a",  karyokinetic 
figures  in  erythrocytes;  the  protoplasm  finely  punctate;  b,  beginning 
karyolysis  in  a  megaloblast;  c,  erythroblasts  with  coarse  granulation  of 
the  protoplasm;  d,  nuclear  remains  (?)  and  line  granulation  of  the 
protoplasm;  e  and  /,  finely  punctate  red  blood-corpuscles;  g,  megalocyte 
with  two  blue  nuclei;  nuclear  remains  (?)  in  the  polychrome  protoplasm. 

(Nothnagel-Lazarus. ) 


PLATE  IX 


Fig.  1. 


0  r  • 

Fig.  2. 


JB^- 


\\:<! 


i  T'    d 


Sf   c 


f-  • 


% 


Fig.  S. 


SPECIAL  BLOOD  PATHOLOGY  279 

The  red  corpuscles  show  marked  variation  in  size  and 
shape  (Plate  IX  and  Fig.  104).  There  is  a  decided 
tendency  to  large  oval  forms,  and  despite  the  abundance 
of  microcytes,  the  average  size  of  the  corpuscles  is  gen- 
erally strikingly  increased.  Polychromatophilia  and 
basophilic  degeneration  are  common.  Nucleated  red 
cells  are  always  present,  although  in  many  instances  care- 
ful search  is  required  to  find  them.  In  the  great  majority 
of  cases  megaloblasts  exceed  normoblasts  in  number. 
This  ratio  constitutes  one  of  the  most  important  points 
in  diagnosis,  since  it  is  practically  unknown  in  other 
diseases.     Blood-plaques  are  diminished. 

The  rare  and  rapidly  fatal  anemia  which  has  been 
described  under  the  name  of  aplastic  anemia  is  probably 
a  variety  of  pernicious  anemia.  Absence  of  any  attempt 
at  blood  regeneration  explains  the  marked  difference  in 
the  blood-picture.  Red  corpuscles  and  hemoglobin  are 
rapidly  diminished  to  an  extreme  degree.  The  color- 
index  is  normal  or  low.  The  leukocyte  count  is  normal 
or  low,  with  relative  increase  of  lymphocytes.  Stained 
smears  show  only  slight  variations  in  size,  shape,  and 
staining  properties  of  the  red  cells.  There  are  no  megalo- 
blasts and  few  or  no  normoblasts. 

(2)  Chlorosis. — The  clinical  symptoms  furnish  the 
most  important  data  for  diagnosis.  The  blood  resembles 
that  of  secondary  anemia  in  many  respects. 

The  most  conspicuous  feature  is  a  decided  decrease  of 
hemoglobin  (down  to  30  or  40  per  cent,  in  marked  cases), 
accompanied  by  a  slight  decrease  in  number  of  red  cor- 
puscles. The  color-index  is  thus  almost  invariably  low, 
the  average  being  about  0.5. 

As  in  pernicious  anemia,  the  leukocytes  are  normal  or 


28o  THE   BLOOD 

decreased  in  number,  with  a  relative  increase  of  lympho- 
cytes. 

In  contrast  to  pernicious  anemia  (and  in  some  degree 
also  to  secondary  anemia)  the  red  cells  are  of  nearly 
uniform  size,  are  uniformly  pale  (Fig,  104),  and  their 
average  diameter  is  somewhat  less  than  normal.  Changes 
in  size,  shape,  and  staining  reactions  occur  only  in  severe 
cases.  Erythroblasts  are  rarely  present.  The  number 
of  plaques  is  generally  decreased. 

(3)  Splenic  Anemia. — This  is  an  obscure  form  of 
anemia  associated  with  great  enlargement  of  the  spleen. 
It  is  probably  a  distinct  entity.  There  is  decided  decrease 
of  hemoglobin  and  red  corpuscles,  with  moderate  leu- 
kopenia and  relative  lymphocytosis.  Osier's  15  cases 
averaged  47  per  cent,  hemoglobin  and  3,336,357  red  cells. 
Stained  films  show  notable  irregularities  in  size,  shape, 
and  staining  properties  only  in  advanced  cases.  Erythro- 
blasts are  uncommon. 

B.    Leukemia 

Except  in  rare  instances,  diagnosis  is  easily  made 
from  the  blood  alone.  Two  types  of  the  disease  are 
commonly  distinguished:  the  myelogenous  and  the  lym- 
phatic. Atypical  and  intermediate  forms  are  not  un- 
common. Pseudoleukemia,  because  of  its  clinical  sim- 
ilarity to  lymphatic  leukemia,  is  generally  described 
along  with  leukemia. 

1.  Myelogenous  Leukemia  (Plate  X).— This  is  usu- 
ally a  chronic  disease,  although  acute  cases  have  been 
described. 

Hemoglobin  and  red  corpuscles  show  decided  decrease. 
The  color-index  is  moderately  low. 


PLATE  X 


Fig.  I. — Blood  in  lymphatic  leukemia;  X  700.     On  the  left,  chronic  form 
of  the  disease;  on  the  right,  acute  form  (courtesy  of  Dr.  W.  P.  Harlow). 


Fig.  2. — Blood  in  splenomyelogenous  leukemia.     Wright's  stain.     X  700 
(photographs  by  the  author). 


SPEaAL   BLOOD  PATHOLOGY  28 1 

Most  striking  is  the  immense  increase  in  number  of 
leukocytes.  The  count  in  ordinary  cases  varies  between 
100,000  and  300,000.  Counts  over  1,000,000  have  been 
met.  During  remissions,  the  leukocyte  count  may  fall 
to  normal. 

While  these  enormous  leukocyte  counts  are  equaled  in 
no  other  disease,  and  approached  only  in  lymphatic 
leukemia  and  extremely  high-grade  leukocytosis,  the 
diagnosis,  particularly  during  remissions,  depends  more 
upon  qualitative  than  quantitative  changes.  Although 
all  varieties  are  increased,  the  characteristic  and  con- 
spicuous cell  is  the  myelocyte.  This  cell  never  appears 
in  normal  blood;  extremely  rarely  in  leukocytosis;  and 
never  abundantly  in  lymphatic  leukemia.  In  myelog- 
enous leukemia  myelocytes  usually  constitute  more  than 
20  per  cent,  of  all  leukocytes.  Da  Costa's  lowest  case 
gave  7  per  cent.  The  neutrophihc  form  is  generally 
much  more  abundant  than  the  eosinophiUc.  Both  show 
considerable  variations  in  size.  Very  constant  also  is  a 
marked  absolute,  and  often  a  relative,  increase  of  eosin- 
ophiles  and  basophiles.  Polymorphonuclear  neutro- 
philes  and  lymphocytes  are  relatively  decreased. 

The  red  cells  show  the  changes  characteristic  of  a 
severe  secondary  anemia,  except  that  nucleated  reds  are 
commonly  abundant;  in  fact,  no  other  disease  gives  so 
many.  They  are  chiefly  of  the  normoblastic  type. 
Megaloblasts  are  uncommon.  Blood-plaques  are  gen- 
erally increased. 

2.  Lymphatic  Leukemia  (Plate  X).— This  form  may 
be  either  acute  or  chronic.  There  is  marked  loss  of 
hemoglobin  and  red  corpuscles.  The  color-index  is 
usually  moderately  low. 


282  THE   BLOOD 

The  leukocyte  count  is  high,  but  lower  than  in  the 
myelogenous  type.  Counts  of  100,000  are  about  the 
average,  but  in  many  cases  are  much  lower.  This  high 
count  is  referable  almost  wholly  to  increase  of  lympho- 
cytes. They  generally  exceed  90  per  cent,  of  the  total 
number.  In  chronic  cases  they  are  chiefly  of  the  small 
variety;  in  acute  cases,  of  the  large  form.  Myelocytes 
are  rare. 

The  red  corpuscles  show  the  changes  usual  in  severe 
secondary  anemia.  Erythroblasts  are  seldom  abundant. 
Blood-plaques  are  decreased. 

3.  Pseudoleukemia  (Hodgkin's  disease)  resembles 
lymphatic  leukemia  in  that  there  is  marked  and  pro- 
gressive enlargement  of  the  lymph-nodes.  There  is, 
however,  no  distinctive  blood-picture.  The  changes  in 
hemoglobin  and  red  cells  resemble  those  of  a  moderate 
symptomatic  anemia,  with  rather  low  color-index.  The 
leukocytes  are  commonly  normal  in  number  and  relative 
proportions. 

4.  Anaemia  Infantum  PseudoJeukaemica. — Under 
this  name  von  Jaksch  described  a  rare  disease  of  infancy, 
the  proper  classification  of  which  is  uncertain.  There  is 
enlargement  of  liver  and  spleen,  and  sometimes  of  lymph- 
nodes,  together  with  the  following  blood  changes:  grave 
anemia  with  deformed  and  degenerated  red  cells  and 
many  erythroblasts  of  both  normoblastic  and  megalo- 
blastic types;  great  increase  in  number  of  leukocytes 
(20,000  to  100,000)  and  great  variations  in  size,  shape, 
and  staining  of  leukocytes,  with  many  atypic  forms,  and 
a  few  myelocytes. 

The  table  on  the  following  page  contrasts  the  distinct- 
ive blood-changes  in  the  more  common  conditions. 


SPECIAI.  BLOOD  PATHOLOGY  283 


n  1 

a-n  5  2  B-       3  «  ;^  5.      o 


a- 

1! 

p 

p 

i 

1 

1 

p-^< 

&. 

I 

S 

P 

3' 

-1 

!>r 

1 

s. 

n 

o-v: 

3 
5_ 

2L 
era' 

a 

En' 

cr 

,'~^ 

p_ 

(D 

a- 

6: 

1 

t3 

3. 

C/3 

i 

P" 

1! 

►1 

0 

P 

c 

M 

-1 

C/1 

>-( 

r-f- 

^ 

0 

?■ 

Ul 

3 

0 

Q 

£=" 

P 

D- 

lA 

P 

in 

P 

3 

3 

0 

0) 

»-t 

-0 

ft 

.  n 

<; 

.  0 

3 

0 

,3^3 

•? 

3* 
0 

Cl 

0 

0 

n 

0^ 

3 

3- CO 

e 

(T) 

rt 

3  y. 

3-S.  c-^ 


crS-  3.      S-™  StS" 


i-^ 


=1-  2.  a.  O      Q  w       hrj 


3  ^ 

3   D.               "."d-                                2-P           o2         3""  "Tj 

11  1^      If  ml  p?  p 

3  "L                                                                ^  2L          n  tr* 

■  &                                              •  Bto     s  ^ 

Km         ^.^                    &^3oS-^Z      r  S 

S          |3                     |3       .^Pg     i§  00 

O 

OQS         MOW         a'P'Onji-.tB         3"P<2L  trl 

o-i"  3""^i-§  S    li  -S  S>o  P    i?  o'5"|  q  M 

=  S  p"  3  S-"v^  2  "  3  P-     p  ^  3  "■  §'3-&.  g  t-* 

^^  S-  i-     ^  c   S^  S  H5^g,<   o  §  "■  a  3    n  g 

t-^l   ^Ti2  3.  S.S      2S      e   sS-s-3"'    o  O 

3 

c 


06  Pi  a  ^  3  2;  o  ffi. -•  3:  ^     oM       ■^ 


^^  ^  "E.P  ^  3  g  w|p  |r  S  -  ^  0.5-  3        O 


3 

CgS'^i  '^3        ^n        3.  fej 

3  3  &"^  o  2.  i2.  i  3  1^^  !2.(2  S 


S-^    P  ':5  S  S   2:  <S  5-  2,  S 


cPs   "ifiicLg;   w^i.^ 


<.^  \^n  3-^  P  P  v: 

uji^^W'ot^EJOQ  ,"3 


P  sT  £: !«  2  3 

P  i  "  5-<^  ^ 
s    •  M         3  '^  P  o -"  "> 

1    fl)  ^S    n         .  O    O 

S-a      33g^3_3 
o^         3--S  P  J5  2  9 


2."S- 

^  3^ 


CHAPTER  IV 

THE  STOMACH 

Laboratory  methods  may  be  applied  to  the  diagnosis 
of  stomach  disorders  in :  I.  Examination  of  the  gastric  con- 
tents removed  with  the  stomach-tube.  II.  Certain  other 
examinations  which  give  information  as  to  the  condition 
of  the  stomach. 

I.  EXAMINATION  OF  THE  GASTRIC  CONTENTS 

Stomach  digestion  consists  mainly  in  the  action  of  pep- 
sin upon  proteins  in  the  presence  of  hydrochloric  acid 
and  in  the  curdling  of  milk  by  rennin.  The  fat-splitting 
ferment,  lipase,  of  the  gastric  juice  has  very  little  activity 
under  normal  conditions  of  acidity. 

Pepsin  and  rennin  are  secreted  by  the  gastric  glands 
as  zymogens — pepsinogen  and  renninogen  respectively 
— which  are  converted  into  pepsin  and  rennin  by  hydro- 
chloric acid.  Hydrochloric  acid  is  secreted  by  certain 
cells  of  the  fundus  glands.  It  at  once  combines  loosely 
with  the  proteins  of  the  food,  forming  acid-metaprotein, 
the  first  step  in  protein  digestion.  Hydrochloric  acid, 
which  is  thus  loosely  combined  with  proteins,  is  called 
"combined"  hydrochloric  acid.  The  acid  which  is  se- 
creted after  the  proteins  present  have  all  been  converted 
into  acid-metaprotein  remains  as  "free"  hydrochloric 
acid,  and,  together  with  pepsin,  continues  the  process  of 
digestion. 

284 


EXAMINATION   OF  THE  GASTRIC  CONTENTS         285 

At  the  height  of  digestion  the  stomach-contents  consist 
essentially  of:  (i)  Water;  (2)  free  hydrochloric  acid; 
(3)  combined  hydrochloric  acid;  (4)  pepsin;  (5)  rennin; 
(6)  mineral  salts,  chiefly  acid  phosphates,  of  no  clinical 
importance;  (7)  particles  of  undigested  and  partly  di- 
gested food;  (8)  various  products  of  digestion  in  solution. 
In  pathologic  conditions  there  may  be  present,  in  addi- 
tion, various  microscopic  structures  and  certain  organic 
acids,  of  which  lactic  acid  is  most  important. 

A  routine  examination  is  conveniently  carried  out  in  the 
following  order: 

(i)  Give  the  patient  a  test-meal  upon  an  empty  stomach, 
washing  the  stomach  previously  if  necessary. 

(2)  At  the  height  of  digestion,  usually  in  one  hour,  remove 
the  contents  of  the  stomach  with  a  stomach-tube. 

(3)  Measure  and  examine  macroscopicaliy. 

(4)  Filter.  A  suction  filter  is  desirable,  and  may  be  neces- 
sary when  much  mucus  is  present. 

(5)  During  filtration,  examine  microscopically  and  make 
qualitative  tests  for — (a)  free  acids;  (b)  free  hydrochloric 
acid;  (c)  lactic  acid. 

(6)  When  sufficient  filtrate  is  obtained,  make  quantitative 
estimations  of — (a)  total  acidity;  (b)  free  hydrochloric  acid; 
(c)  combined  hydrochloric  acid  (if  ne'cessary). 

(7)  Make  whatever  additional  tests  seem  desirable,  as  for 
blood,  pepsin,  or  rennin. 

A.     Obtaining  the  Contents 

■  Gastric  juice  is  secreted  continuously,  but  quantities 
suflSciently  large  for  examination  are  not  usually  obtain- 
able from  the  fasting  stomach.  In  clinical  work,  there- 
fore, it  is  desirable  to  stimulate  secretion  with  food — 


286  THE   STOMACH 

which  is  the  natural  and  most  efficient  stimulus — before 
attempting  to  collect  the  gastric  fluid.  Different  foods 
stimulate  secretion  to  different  degrees,  hence  for  the 
sake  of  uniform  results  certain  standard  "test-meals" 
have  been  adopted.  Those  mentioned  here  give  prac- 
tically the  same  results. 

1 .  Test=meals. — It  is  customary  to  give  the  test-meal 
in  the  morning,  since  the  stomach  is  most  apt  to  be 
empty  at  that  time.  If  it  be  suspected  that  the  stomach 
will  not  be  empty,  it  should  be  washed  out  with  water 
the  evening  before. 

(i)  Ewald's  test-breakfast  consists  of  a  roll  (or  two 
slices  of  bread),  without  butter,  and  two  small  cups  (300 
to  400  c.c.)  of  water,  or  weak  tea,  without  cream  or  sugar. 
It  should  be  well  masticated.  The  contents  of  the 
stomach  are  to  be  removed  one  hour  afterward,  counting 
from  the  beginning,  not  the  end  of  the  meal.  This 
test-meal  has  long  been  used  for  routine  examinations. 
Its  disadvantage  is  that  it  introduces,  with  the  bread,  a 
variable  amount  of  lactic  acid  and  numerous  yeast-cells. 
This  source  of  error  may  be  eliminated  by  substituting 
a  shredded  whole-wheat  biscuit  for  the  roll.  The 
shredded  wheat  test-meal  is  now  widely  used  and  is 
probably  the  most  satisfactory  for  general  purposes. 

(2)  Boas'  test-breakfast  consists  of  a  tablespoonful 
of  rolled  oats  in  a  quart  of  water,  boiled  to  one  pint,  with 
a  pinch  of  salt  added.  It  should  be  withdrawn  in  forty- 
five  minutes  to  one  hour.  This  meal  does  not  contain 
lactic  acid,  and  is  usually  given  when  the  detection  of 
lactic  acid  is  important,  as  in  suspected  gastric  cancer. 
The  stomach  should  always  be  washed  with  water  the 
evening  previous. 


EXAMINATION   OF  THE   GASTRIC  CONTENTS  287 

2.  Withdrawal  of  the  Contents. — The  Boas  stomach- 
tube,  with  bulb,  is  probably  the  most  satisfactory  form. 
It  should  be  of  rather  large  caHber,  and  have  an  opening  in 
the  tip  and  one  or  two  in  the  side  near  the  tip.  When  not 
in  use  it  should  be  kept  in  a  vessel  of  borax  solution,  and 
should  be  well  washed  in  hot  water  both  before  and  after 
using. 

It  is  important  confidently  to  assure  the  patient  that 
introduction  of  the  tube  cannot  possibly  harm  him;  and 
that,  if  he  can  control  the  spasm  of  his  throat,  he  will 
experience  very  little  choking  sensation.  When  patients 
are  very  nervous  it  is  well  to  spray  the  throat  with  cocain 
solution. 

The  tube  should  be  dipped  in  warm  water  just  before 
using:  the  use  of  glycerin  or  other  lubricant  is  undesir- 
able. With  the  patient  seated  upon  a  chair,  his  cloth- 
ing protected  by  towels  or  a  large  apron,  and  his  head 
tilted  forward,  the  tip  of  the  tube,  held  as  one  would  a 
pen,  is  introduced  far  back  into  the  pharynx.  He  is  then 
urged  to  swallow,  and  the  tube  is  pushed  boldly  into  the 
esophagus  until  the  ring  upon  it  reaches  the  incisor  teeth, 
thus  indicating  that  the  tip  is  in  the  stomach.  If,  now, 
the  patient  cough  or  strain  as  if  at  stool,  the  contents  of 
the  stomach  will  usually  be  forced  out  through  the  tube. 
Should  it  fail,  the  fluid  can  generally  be  pumped  out  by 
alternate  compression  of  the  tube  and  the  bulb.  If 
unsuccessful  at  first,  the  attempts  should  be  repeated 
with  the  tube  pushed  a  little  further  in,  or  withdrawn  a 
few  inches,  since  the  distance  to  the  stomach  is  not  the 
same  in  all  cases.  The  tube  may  become  clogged  with 
pieces  of  food,  in  which  case  it  must  be  withdrawn, 
cleaned,  and  reintroduced.    If,  after  all  efforts,  no  fluid 


288  THE  STOMACH 

is  obtained,  another  test-meal  should  be  given  and  with- 
drawn in  forty-five  minutes. 

As  the  tube  is  removed,  it  should  be  pinched  between 
the  fingers  so  as  to  save  any  fluid  that  may  be  in  it. 

The  stomach-tube  must  be  used  with  great  care,  or  not 
at  all,  in  cases  of  gastric  ulcer,  aneurysm,  uncompensated 
heart  disease,  and  marked  arteriosclerosis.  Except  in 
gastric  ulcer,  the  danger  lies  in  the  retching  produced,  and 
the  tube  can  safely  be  used  if  the  patient  takes  it  easily. 

B.  Physical  Examination 
Under  normal  conditions  30  to  50  c.c.  of  fluid  can  be 
obtained  one  hour  after  administering  Ewald's  breakfast. 
More  than  60  c.c.  points  to  motor  insufficiency;  less 
than  20  c.c,  to  too  rapid  emptying  of  the  stomach,  or 
else  to  incomplete  removal.  Upon  standing,  it  separates 
into  two  layers,  the  lower  consisting  of  particles  of  food, 
the  upper  of  an  almost  clear,  faintly  yellow  fluid.  The 
extent  to  which  digestion  has  taken  place  can  be  roughly 
judged  from  the  appearance  of  the  food-particles. 

The  reaction  is  frankly  acid  in  health  and  in  nearly  all 
pathologic  conditions.  It  may  be  neutral  or  slightly 
alkaline  in  some  cases  of  gastric  cancer  and  marked 
chronic  gastritis,  or  when  contaminated  by  a  consider- 
able amount  of  saliva. 

A  small  amount  of  mucus  is  present  normally.  Large 
amounts,  when  the  gastric  contents  are  obtained  with 
the  tube  and  not  vomited,  point  to  chronic  gastritis. 
Mucus  is  recognized  from  its  characteristic  slimy  appear- 
ance when  the  fluid  is  poured  from  one  vessel  into  another. 
It  is  more  frequently  seen  in  stomach  washings  than  in 
the  fluid  removed  after  a  test-meal. 


EXAMINATION   OF  THE   GASTRIC  CONTENTS  289 

A  trace  of  bile  may  be  present  as  a  result  of  excessive 
straining  while  the  tube  is  in  the  stomach.  Large 
amounts  are  very  farely  found,  and  generally  point  to 
obstruction  in  the  duodenum.  Bile  produces  a  yellowish 
or  greenish  discoloration  of  the  fluid. 

Blood  is  often  recognized  by  simple  inspection,  but 
more  frequently  requires  a  chemic  test.  It  is  bright  red 
when  very  fresh,  and  dark,  resembling  coffee-grounds, 
when  older.  Vomiting  of  blood,  or  hematemesis,  may  be 
mistaken  for  pulmonary  hemorrhage,  or  hemoptysis.  In 
the  former  the  fluid  is  acid  in  reaction  and  usually  dark 
red  or  brown  in  color  and  clotted,  while  in  hemoptysis 
it  is  brighter  red,  frothy,  alkaline,  and  usually  mixed 
with  a  variable  amount  of  mucus. 

Particles  of  food  eaten  hours  or  even  days  previously 
may  be  found,  and  indicate  deficient  motor  power. 

Search  should  always  be  made  for  bits  of  tissue  from 
the  gastric  mucous  membrane  or  new  growths.  These, 
when  examined  by  a  pathologist,  will  sometimes  render 
the  diagnosis  clear. 

C.  Chemic  Examination 
A  routine  chemic  examination  of  the  gastric  contents 
involves  qualitative  tests  for  free  acids,  free  hydrochloric 
acid,  and  organic  acids,  and  quantitative  estimations  of 
total  acidity,  free  hydrochloric  acid,  and  sometimes 
combined  hydrochloric  acid.  Other  tests  are  applied 
when  indicated. 

*  I.  Qualitative  Tests.— (i)  Free  Acids.— The  pres- 
ence or  absence  of  free  acids,  without  reference  to  the 
kind,  is  easily  determined  by  means  of  Congo-red, 
although  the  test  is  not  much  used  in  practice. 

19 


290  THE   STOMACH 

Congo-red  Test. — Take  a  few  drops  of  a  strong  alcoholic 
solution  of  Congo-red  in  a  test-tube,  dilute  with  water  to  a 
strong  red  color,  and  add  a  few  cubic  centimeters  of  filtered 
gastric  juice.  The  appearance  of  a  blue  color  shows  the 
presence  of  some  free  acid  (Plate  XI,  B,  B').  Since  the  test 
is  more  sensitive  to  mineral  than  to  organic  acids,  a  marked 
reaction  points  to  the  presence  of  free  hydrochloric  acid. 

Thick  filter-paper  soaked  in  Congo-red  solution,  dried,  and 
cut  into  strips  may  be  used,  but  the  test  is  much  less  delicate 
when  thus  applied. 

(2)  Free  Hydrochloric  Acid. — In  addition  to  its  diges- 
tive function,  free  hydrochloric  acid  is  an  efficient  anti- 
septic. It  prevents  or  retards  fermentation  and  lactic- 
acid  formation,  and  is  an  important  means  of  protection 
against  the  entrance  of  pathogenic  organisms  into  the 
body.     It  is  never  absent  in  health. 

Amidobenzol  Test. — To  a  little  of  the  filtered  gastric  juice 
in  a  test-tube,  or  to  several  drops  in  a  porcelain  dish,  add  a 
drop  of  0.5  per  cent,  alcoholic  solution  of  dimethylamido- 
azobenzol.  In  the  presence  of  free  hydrochloric  acid  there 
will  at  once  appear  a  cherry-red  color,  varying  in  intensity 
with  the  amount  of  acid  (Plate  XII,  C).  This  test  is  very 
delicate;  but,  unfortunately,  organic  acids,  when  present  in 
large  amounts  (above  0.5  per  cent.),  give  a  similar  reaction. 

Boas'  Test. — This  test  is  less  delicate  than  the  preceding, 
but  is  more  reliable,  since  it  reacts  only  to  free  hydrochloric 
acid. 

In  a  porcelain  dish  mix  a  few  drops  of  the  gastric  juice  and 
the  reagent,  and  slowly  evaporate  to  dryness  over  a  flame, 
taking  care  not  to  scorch.  The  appearance  of  a  rose-red  color, 
which  fades  upon  cooling,  shows  the  presence  of  free  hydro- 
chloric acid  (Plate  XI,  i). 


PLATE  XI 


A,  Uffelmann's  reagent;  A',  A  after  the  addition  of  gastric  fluid 
containing  lactic  acid;  B,  water  to  which  three  drops  of  Congo-red 
solution  have  been  added;  B',  change  induced  in  B  when  gastric  fluid 
containing  free  hydrochloric  acid  is  added  (Boston). 


I,  Resorcin-test  for  free  hydrochloric  acid;  2,  Glinzburg's  test  for  hydro- 
chloric acid  (Boston). 


EXAMINATION   OF  THE   GASTRIC  CONTENTS  29 1 

Boas'  reagent  consists  of  5  gm.  resublimed  resorcinol,  and 
3  gm.  cane-sugar,  in  100  c.c.  alcohol.  The  solution  keeps 
well,  which,  from  the  practitioner's  view-point,  makes  it 
preferable  to  Giinzburg's  phloroglucin-vanillin  reagent  (phlo- 
roglucin,  2  gm.;  vanillin,  i  gm.;  absolute  alcohol,  30  c.c). 
The  latter  is  just  as  delicate,  is  applied  in  the  same  way,  and 
gives  a  sharper  reaction  (Plate  XI,  2),  but  is  unstable. 

(3)  Organic  Acids. — Lactic  acid  is  the  most  common, 
and  is  taken  as  the  type  of  the  organic  acids  which  appear 
in  the  stomach-contents.  It  is  a  product  of  bacterial 
activity.  Acetic  and  butyric  acids  are  sometimes 
present.  Their  formation  is  closely  connected  with  that 
of  lactic  acid,  and  they  are  rarely  tested  for.  When 
abundant,  they  may  be  recognized  by  their  odor  upon 
heating. 

Lactic  acid  is  never  present  at  the  height  of  digestion 
in  health.  Although  usually  present  early  in  digestion, 
it  disappears  when  free  hydrochloric  acid  begins  to 
appear.  Small  amounts  may  be  introduced  with  the 
food.  Pathologically,  small  amounts  may  be  present 
whenever  there  is  stagnation  of  the  gastric  contents  with 
deficient  hydrochloric  acid,  as  in  many  cases  of  dilatation 
of  the  stomach  and  chronic  gastritis.  The  presence  of 
notable  amounts  of  lactic  acid  (more  than  o.i  per  cent, 
by  Strauss'  test)  is  strongly  suggestive  of  gastric  can- 
cer, and  is  probably  the  most  valuable  single  symp- 
tom of  the  disease. 

As  already  stated,  the  Ewald  test-breakfast  introduces 
a  small  amount  of  lactic  acid,  but  rarely  enough  to  re- 
spond to  the  tests  given  here.  In  every  case,  however, 
in  which  its  detection  is  important,  the  shredded- wheat 
biscuit   or   Boas'   test-breakfast   should  be   given,   the 


292  THE   STOMACH 

stomach  having  been  thoroughly  washed  the  evening 
before. 

Ufifelmann's  Test  for  Lactic  Acid. — Thoroughly  shake 
up  5  c.c.  of  filtered  stomach  fluid  with  50  c.c.  of  ether  for  at 
least  ten  minutes.  Collect  the  ether  and  evaporate  over  a 
water-bath.  Dissolve  the  residue  in  5  c.c.  water  and  test 
with  Ufifelmann's  reagent  as  follows: 

In  a  test-tube  mix  3  drops  concentrated  solution  of 
phenol  and  3  drops  saturated  aqueous  solution  of  ferric 
chlorid.  Add  water  until  the  mixture  assumes  an  amethyst- 
blue  color.  To  this  add  the  solution  to  be  tested.  The 
appearance  of  a  canary-yellow  color  indicates  the  presence  of 
lactic  acid  (Plate  XI,  A,  A'). 

Uffelmann's  test  may  be  applied  directly  to  the  stomach- 
contents  without  extracting  with  ether,  but  is  then  neither 
sensitive  nor  reliable,  because  of  the  phosphates,  sugars,  and 
other  interfering  substances  which  may  be  present. 

Kelling's  Test  {Simofis  Modification). — This  is  much  more 
satisfactory  than  Ufifelmann's.  To  a  test-tube  of  distilled 
water  add  sufficient  ferric  chlorid  solution  to  give  a  faint 
yellowish  tinge.  Pour  half  of  this  into  a  second  test-tube  to 
serve  as  a  control.  To  the  other  add  a  small  amount  of  the 
gastric  juice.  Lactic  acid  gives  a  distinct  yellow  color  which 
is  readily  recognized  by  comparison  with  the  control. 

Strauss'  Test  for  Lactic  Acid. — This  is  a  good  test  for 
clinical  work,  since  it  gives  a  rough  idea  of  the  quantity 
present  and  is  not  sufficiently  sensitive  to  respond  to  the 
traces  of  lactic  acid  which  some  test-meals  introduce.  Strauss' 
instrument  (Fig.  105)  is  essentially  a  separating  funnel  with 
a  mark  at  5  c.c.  and  one  at  25  c.c.  Fill  to  the  5  c.c.  mark 
with  filtered  stomach  fluid,  and  to  the  25  c.c.  mark  with  ether. 
Shake  thoroughly  for  ten  or  fifteen  minutes,  let  stand  until  the 
ether  separates,  and  then,  by  opening  the  stop-cock,  allow  the 
liquid  to  run  out  to  the  5  c.c.  mark.     Fill  to  the  25  c.c.  mark 


EXAMINATION   OF  THE   GASTRIC  CONTENTS 


293 


with  water,  and  add  two  drops  of  tincture  of  ferric  chlorid 
diluted  I  :  ID.  Shake  gently.  If  o.i  per  cent,  or  more  lactic 
acid  be  present,  the  water  will  assume  a 
strong  greenish-yellow  color.  A  slight 
tinge  will  appear  with  0.05  per  cent. 

(4)  Pepsin  and  Pepsinogen. — Pep- 
sinogen itself  has  no  digestive  power. 
It  is  secreted  by  the  gastric  glands,  and 
is  transformed  into  pepsin  by  the  ac- 
tion of  a  free  acid.  Although  pepsin 
digests  proteins  best  in  the  presence 
of  free  hydrochloric  acid,  it  has  a  slight 
digestive  activity  in  the  presence  of 
organic  or  combined  hydrochloric  acids. 

The  amount  is  not  influenced  by 
neuroses  or  circulatory  disturbances. 
Absence  or  marked  diminution,  there- 
fore, indicates  organic  disease  of  the 
stomach.  It  is  an  important  point  in 
diagnosis  between  functional  and  or- 
ganic conditions.  Pepsin  is  rarely  or  never  absent  in 
the  presence  of  free  hydrochloric  acid. 


Fig.  105. — Separatory 
funnel  for  Strauss'  lac- 
tic acid  test  (Sahli). 


Test  for  Pepsin  and  Pepsinogen. — With  a  cork-borer  cut 
small  cylinders  from  the  coagulated  white  of  an  egg,  and  cut 
these  into  discs  of  uniform  size.  The  egg  should  be  cooked 
very  slowly,  preferably  over  a  water-bath,  so  that  the  white 
may  be  readily  digestible.  The  discs  may  be  preserved  in 
glycerin,  but  must  be  washed  in  water  before  using. 

Place  a  disc  in  each  of  three  test-tubes. 

Into  tube  No.  i  put  10  c.c.  distilled  water,  5  grains  pepsin, 
U.  S.  P.,  and  3  drops  of  the  official  dilute  hydrochloric  acid. 


294  THE  STOMACH 

Into  tube  No.  2  put  10  c.c.  filtered  gastric  juice. 

Into  tube  No.  3  put  10  c.c.  filtered  gastric  jUice  and  3  drops 
dilute  hydrochloric  acid. 

Place  the  tubes  in  an  incubator  or  warm  water  for  three 
hours  or  longer.  At  intervals,  observe  the  extent  to  which 
the  egg-albumen  has  been  digested.  This  is  recognized  by 
the  depth  to  which  the  disc  has  become  translucent. 

Tube  No.  I  is  used  for  comparison,  and  should  show  the 
effect  of  normal  gastric  juice. 

Digestion  of  the  egg  in  tube  No.  2  indicates  the  presence  of 
both  pepsin  and  free  hydrochloric  acid. 

When  digestion  fails  in  tube  No.  2  and  occurs  in  No.  3, 
pepsinogen  is  present,  having  been  transformed  into  pepsin  by 
the  hydrochloric  acid  added.  Should  digestion  fail  in,  this 
tube,  both  pepsin  and  pepsinogen  are  absent. 

(5)  Rennin  and  Renninogen. — Rennin  is  the  milk- 
curdling  ferment  of  the  gastric  juice.  It  is  derived  from 
reiminogen  through  the  action  of  hydrochloric  acid. 
Lime  salts  also  possess  the  power  of  transforming  rennin- 
ogen into  the  active  ferment. 

Deficiency  of  rennin  has  the  same  significance  as 
deficiency  of  pepsin,  and  is  more  easily  recognized. 
Since  the  two  enzj-mes  are  almost  invariably  present  or 
absent  together,  the  test  for  rennin  serves  also  as  a  test 
for  pepsin. 

Test  for  Rennin. — Neutralize  5  c.c.  filtered  gastric  juice 
with  very  dilute  sodium  hydroxid  solution;  add  5  c.c.  fresh 
milk,  and  place  in  an  incubator  or  in  a  vessel  of  water  at 
about  104°  F.  Coagulation  of  the  milk  in  ten  to  fifteen 
minutes  shows  a  normal  amount  of  rennin.  Delayed  coagu- 
lation denotes  a  less  amount. 


EXAMINATION   OF  THE   GASTRIC  CONTENTS  295 

Test  for  Renninogen. — To  5  c.c.  neutralized  gastric  Juice 
add  2  c.c.  of  I  per  cent,  calcium  chlorid  solution  and  5  c.c. 
fresh  milk,  and  place  in  an  incubator.  If  coagulation  occurs, 
renninogen  is  present. 

(6)  Blood. — Blood  is  present  in  the  vomitus  in  a 
great  variety  of  conditions.  When  found  in  the  fluid 
removed  after  a  test-meal,  it  commonly  points  toward 
ulcer  or  carcinoma.  Blood  can  be  detected  in  nearly 
one-half  of  the  cases  of  gastric  cancer.  The  presence  of 
swallowed  blood  must  be  excluded. 

Test  for  Blood  in  Stomach-contents. — To  10  c.c.  of  the 

fluid  add  a  few  cubic  centimeters  of  glacial  acetic  acid  and 
shake  the  mixture  thoroughly  with  an  equal  volume  of  ether. 
Separate  the  ether  and  apply  to  it  the  guaiac  test  (p.  125) ;  or 
evaporate  and  apply  the  hemin  test  (p.  274)  to  the  residue. 
When  brown  particles  are  present  in  the  fluid,  the  hemin  test 
should  be  applied  directly  to  them. 

2.  Quantitative  Tests.— (i)  Total  Acidity.— The  acid- 
reacting  substances  which  contribute  to  the  total  acidity 
are  free  hydrochloric  acid,  combined  hydrochloric  acid, 
acid  salts,  mostly  phosphates,  and,  in  some  pathologic 
conditions,  the  organic  acids.  The  total  acidity  is 
normally  about  50  to  75  degrees  (see  method  below)  or, 
when  estimated  as  hydrochloric  acid,  about  0.2  to  0.3 
per  cent. 

Tbpfer's  Method  for  Total  Acidity. — In  an  evaporating 
dish  or  small  beaker  (an  "  after-dinner  "  coffee-cup  is  a  very 
convenient  substitute)  take  10  c.c.  filtered  stomach-contents 
and  add  three  or  four  drops  of  the  indicator,  a  i  per  cent, 
alcoholic  solution  of  phenolphthalein.    When  the  quantity  of 


296  THE  STOMACH 

stomach  fluid  is  small,  5  c.c.  may  be  used,  but  results  are  less 
accurate  than  with  a  larger  amount.  Add  decinormal  solution 
of  sodium  hydroxid  drop  by  drop  from  a  buret,  until  the  fluid 
assumes  a  rose-red  color  which  does  not  become  deeper  upon 
addition  of  another  drop  (Plate  XII,  A,  A').  When  this  point 
is  reached,  all  the  acid  has  been  neutralized.  The  end  reaction 
will  be  sharper  if  the  fluid  be  saturated  with  sodium  chlorid. 
A  sheet  of  white  paper  beneath  the  beaker  facilitates  recog- 
nition of  the  color  change. 

In  clinical  work  the  amount  of  acidity  is  expressed  by  the 
number  of  cubic  centimeters  of  the  decinormal  sodium  hy- 
droxid solution  which  would  be  required  to  neutralize  100  c.c. 
of  the  gastric  juice,  each  cubic  centimeter  representing  one 
deforce  of  acidity.  Hence  multi]:)ly  the  number  of  cubic  centi- 
meters of  decinormal  solution  required  to  neutralize  the  10  c.c. 
of  stomach  fluid  by  ten.  This  gives  the  number  of  degrees  of 
acidity.  The  amount  may  be  expressed  in  terms  of  hydro- 
chloric acid,  if  one  remember  that  each  degree  is  equivalent  to 
0.00365  per  cent,  hydrochloric  acid.  Some  one  suggests  that 
this  is  the  number  of  days  in  the  year,  the  last  figure,  5,  in- 
dicating the  number  of  decimal  places. 

Example. — Suppose  that  7  c.c.  of  decinormal  solution  were 
required  to  bring  about  the  end  reaction  in  10  c.c.  gastric 
juice;  then  7  X  10  =  70  deforces  of  acidity;  and,  expressed  in 
terms  of  hydrochloric  acid,  70  X  0.00365  =  0.255  P^''  c^^'- 

Preparation  of  decinormal  solution  is  described  in  text- 
books on  chemistry.  The  practitioner  will  find  it  best  to 
have  them  made  by  a  chemist,  or  to  purchase  from  a  chemic 
supply  house.  Preparation  of  an  approximately  decinormal 
solution  is  described  on  page  436. 

(2)  Hydrochloric  Acid. — After  the  Ewald  and  Boas 
test-breakfasts  the  amount  of  free  hydrochloric  acid 
varies  normally  between  25  and  50  degrees,  or  about  o.i 


EXAMINATION   OF  THE  GASTRIC  CONTENTS  297 

to  0.2  per  cent.  In  disease  it  may  go  considerably 
higher  or  may  be  absent  altogether. 

When  the  amount  of  free  hydrochloric  acid  is  normal, 
organic  disease  of  the  stomach  probably  does  not  exist. 

Increase  of  free  hydrochloric  acid  above  50  degrees 
(hyperchlorhydria)  generally  indicates  a  neurosis,  but  also 
occurs  in  most  cases  of  gastric  ulcer  and  beginning 
chronic  gastritis. 

Decrease  of  free  hydrochloric  acid  below  25  degrees 
{hy pochlorhydria)  occurs  in  some  neuroses,  chronic  gas- 
tritis, early  carcinoma,  and  most  conditions  associated 
with  general  systemic  depression.  Marked  variation  in 
the  amount  at  successive  examinations  strongly  suggests 
a  neurosis.  Too  low  values  are  often  obtained  at  the 
first  examination,  the  patient's  dread  of  the  introduction 
of  the  tube  probably  inhibiting  secretion. 

Absence  of  free  hydrochloric  acid  (achlorhydria)  occurs 
in  most  cases  of  gastric  cancer  and  far-advanced  chronic 
gastritis,  in  many  cases  of  pernicious  anemia,  and  some- 
times in  hysteria  and  pulmonary  tuberculosis. 

The  presence  of  free  hydrochloric  acid  presupposes  a 
normal  amount  of  combined  hydrochloric  acid,  hence  the 
combined  need  not  be  estimated  when  the  free  acid  has 
been  found.  When,  however,  free  hydrochloric  acid  is 
absent,  it  is  important  to  know  whether  any  acid  is 
secreted,  and  an  estimation  of  the  combined  acid  then 
becomes  of  great  value.  The  normal  average  after  an 
Ewald  breakfast  is  about  10  to  15  degrees,  the  quantity 
depending  upon  the  amount  of  protein  in  the  test-meal. 

Topfer's    Method    for    Free    Hydrochloric    Acid. — In    a 

beaker  take  10  c.c.  filtered  stomach  fluid  and  add  4  drops 


298  THE  STOMACH 

of  the  indicator,  a  0.5  per  cent,  alcoholic  solution  of  dimethyl- 
amido-azobenzol.  A  red  color  instantly  appears  if  free  hydro- 
chloric acid  be  present.  Add  decinormal  sodium  hydroxid 
solution,  drop  by  drop  from  a  buret,  until  the  last  trace  of  red 
just  disappears,  and  a  canary-yellow  color  takes  its  place  (Plate 
XII,  C,  C')-  Read  off  the  number  of  cubic  centimeters  of 
decinormal  solution  added,  and  calculate  the  degrees,  or 
percentage  of  free  hydrochloric  acid,  as  in  Topfer's  method 
for  total  acidity. 

When  it  is  impossible  to  obtain  sufficient  fluid  for  all  the 
tests,  it  will  be  found  convenient  to  estimate  the  free  hydro- 
chloric acid  and  total  acidity  in  the  same  portion.  After 
finding  the  free  hydrochloric  acid  as  just  described,  add  4 
drops  phenolphthalein  solution,  and  continue  the  titration. 
The  amount  of  decinormal  solution  used  in  both  titrations 
indicates  the  total  acidity. 

Topfer's  Method  for  Combined  Hydrochloric  Acid. — 
In  a  beaker  take  10  c.c.  filtered  gastric  juice  and  add  4 
drops  of  the  indicator,  a  i  per  cent,  aqueous  solution  of  sodium 
alizarin  sulphonate.  Titrate  with  decinormal  sodium  hy- 
droxid until  the  appearance  of  a  bluish-violet  color  which  does 
not  become  deeper  upon  addition  of  another  drop  (Plate  XII, 
B,  B').  It  is  difficult,  without  practice,  to  determine  when 
the  right  color  has  been  reached.  A  reddish- violet  appears 
first.  The  shade  which  denotes  the  end  reaction  can  be 
produced  by  adding  2  or  3  drops  of  the  indicator  to  5  c.c. 
of  I  per  cent,  sodium  carbonate  solution. 

Calculate  the  number  of  cubic  centimeters  of  decinormal 
solution  which  would  be  required  for  100  c.c.  of  stomach  fluid. 
This  gives,  in  degrees,  all  the  acidity  except  the  combined  hydro- 
chloric acid.  The  combined  hydrochloric  acid  is  then  found 
by  deducting  this  amount  from  the  total  acidity,  which  has 
been  previously  determined. 

Example. — Suppose  that  5  c.c.  of  decinormal  solution  were 
required  to  produce  the  purple  color  in  10  c.c.  gastric  juice; 


PLATE  XII 


fv_ 


r 


^ 


ffl 


v 


K- 


< 


A,  Gastric  fluid  to  which  a  i  per  cent,  solution  of  phenolphthalein 
has  been  added;  B,  gastric  fluid  to  which  a  i  per  cent,  solution  of  alizarin 
has  been  added;  C,  gastric  fluid  to  which  a  0.5  per  cent,  solution  of 
dimethylamido-azobenzol  has  been  added;  A',  .'\  after  titration  with  a 
decinormal  solution  of  sodium  hydroxid;  B',  B  after  titration  with  a 
decinormal  solution  of  sodium  hydroxid;  C,  C  after  titration  with  a 
decinormal  solution  of  sodium  hydroxid  (Boston). 


EXAMINATION   OF  THE   GASTRIC   CONTENTS  299 

then  5  X  10  =  50  =  all  the  acidity  except  combined  hydrochloric 
acid.  Suppose,  now,  that  the  total  acidity  has  already  been 
found  to  be  70  degrees;  then  70—  50=  20  degrees  of  combined 
hydrochloric  acid;  and  20X0.00365  =  0.073  per  cent. 

When  free  hydrochloric  acid  is  absent,  it  is  probably 
more  helpful  to  estimate  the  acid  deficit  than  the  com- 
bined hydrochloric  acid.  The  acid  deficit  shows  how 
far  the  acid  secreted  by  the  stomach  falls  short  of  satu- 
rating the  protein  (and  bases)  of  the  meal.  It  repre- 
sents the  amount  of  hydrochloric  acid  which  must  be 
added  to  the  fluid  before  a  test  for  free  hydrochloric 
acid  can  be  obtained.  It  is  determined  by  titrating 
with        hydrochloric  acid,  using  dimethyl-amido-azo- 

benzol  as  indicator,  until  the  fluid  assumes  a  red  color. 
The  amount  of  deficit  is  expressed  by  the  number  of 
cubic  centimeters  of  the  decinormal  solution  required 
for  100  c.c.  of  the  stomach  fluid. 

(3)  Organic  Acids. — There  is  no  simple  direct  quan- 
titative method.  After  the  total  acidity  has  been  deter- 
mined, organic  acids  may  be  removed  from  another 
portion  of  the  gastric  filtrate  by  shaking  thoroughly 
with  an  equal  volume  of  neutral  ether,  allowing  the 
fluids  to  separate,  and  repeating  this  process  until  the 
gastric  fluid  has  been  extracted  with  eight  or  ten  times 
its  volume  of  ether.  The  total  acidity  is  then  deter- 
mined, and  the  difference  between  the  two  determinations 
indicates  the  amount  of  organic  acids. 

(4)  Pepsin. — No  direct  method  is  avaliable.  The 
following  is  sufficient  for  clinical  purposes: 

Hammerschlag's  Method. — To  the  white  of  an  egg  add 
twelve  times  its  volume  of  0.4  per  cent,  hydrochloric  acid 


300  THE   STOMACH 

(dilute  hydrochloric  acid,  U.  S.  P.,  4  c.c;  water,  96  c.c), 
mix  well,  and  filter.  This  gives  a  i  per  cent,  egg-albumen 
solution.  Take  10  c.c.  of  this  solution  in  each  of  three  tubes 
or  beakers.  To  .1  add  5  c.c.  gastric  juice;  to  B^  5  c.c.  water 
with  0.5  gm.  pepsin;  to  C,  5  c.c.  water  only.  Place  in  an 
incubator  for  an  hour  and  then  determine  the  amount  of 
albumin  in  each  mixture  by  Esbach's  method.  Tube  C  shows 
the  amount  of  albumin  in  the  test-solution.  The  difference 
betwetnC  and  B  indicates  the  amountof  albumin  which  would 
be  digested  by  normal  gastric  juice.  The  difference  between 
C  and  .1  gives  the  albumin  which  is  digested  by  the  fluid 
under  examination.  Schiitz  has  shown  that  the  amounts  of 
pepsin  in  two  fluids  are  proportionate  to  the  squares  of  the 
products  of  digestion.  Thus,  if  the  amounts  of  albumin  di- 
gested in  tubes  A  and  B  are  to  each  other  as  2  is  to  4,  the 
amounts  of  pepsin  are  to  each  other  as  4  is  to  16. 

Certain  sources  of  error  can  be  eliminated  by  diluting  the 
gastric  juice  several  times  before  testing.  The  most  import- 
ant of  these  are  that  the  law  of  Schiitz  holds  good  only  for 
comparatively  dilute  solutions,  and  that  the  products  of 
peptic  activity  inhibit  digestion. 

Mett's  method  is  generally  preferred  to  the  preceding. 
Put  three  or  four  Mett's  tubes  about  2  cm.  long  into  a  small 
beaker  with  diluted  gastric  juice  (i  c.c.  of  the  filtrate  plus  15 
c.c.  twentieth-normal  hydrochloric  acid).  Place  in  an  incu- 
bator for  twenty-four  hours,  and  then  measure  as  accurately 
as  possible  the  column  which  has  been  digested,  using  a  milli- 
meter scale  and  a  hand  lens  or,  better,  a  low  power  of  the 
microscope  and  an  eye-piece  micrometer.  Square  the  aver- 
age length  of  this  column  (law  of  Schiitz)  and  multiply  by 
the  degree  of  dilution,  16.  The  maximum  figure  obtained  in 
this  way  is  256,  representing  a  digested  column  of  4  mm. 

Prepare  Mett's  tubes  as  follows: 

Beat  up  slightly  the  whites  of  one  or  two  eggs  and  filter. 
Pour  into  a  wide  test-tube  and  stand  in  this  a  number  of 


EXAMINATION   OF  THE   GASTRIC  CONTENTS 


301 


^'r>/./ 


capillary  glass  tubes,  i  to  £;  mm.  'kT  ^ia^neter.    'IVJj^n  the 
tubes  are  filled,  plug  their  ends  -vti^JV )>r/e^4  crumbs, /a,pd  coag- 
ulate the  albumin  by  heating  in  water  jtistsKort  of  b6lli/igf,//r. 
Dip  the  ends  of  the  tube  in  melted  paraffin  and  preserv^^n^tjl ,  ^ 
needed.     Bubbles,  if  present,  will  probably  disappear  in  a  " 
few  days.     When  wanted  for  use,  cut  the  tubes  into  lengths 
of  about  2  cm.    Discard  any  in  which  the  albumin  has  sep- 
arated from  the  wall. 


D.   Microscopic  Examination 

A  drop  of  unfiltered  stomach-contents  is  placed  upon 
a  slide,  covered  with  a  cover-glass,  and  examined  with 
the  16  mm.  and  4  mm.  objectives.     A  drop  of  Lugol's 


Fig.  io6.^GeneraI  view  of  the  gastric  contents:  a.  Squamous  epithelial  cells  from 
esophagus  knd  mouth;  b,  leukocytes;  c,  cylindric  epithelial  cells;  d,  muscle-fibers;  e,  fat- 
droplets  and  fat-crystals;  /,  starch-granules;  g,  chlorophyl-containing  vegetable  mat- 
ters; k,  vegetable  spirals;  i,  bacteria;  k,  sarcinae;  I,  budding  (yeast)  fungi  (Jakob). 


solution  allowed  to  run  under  the  cover  will  aid  in  dis- 
tinguishing the  various  structures. 

Under  normal  conditions  little  is  to  be  seen  except 
■  great  numbers  of  starch-granules,  with  an  occasional 


302  THE   STOMACH 

epithelial  cell,  yeast-cell,  or  bacterium.  Starch-granules 
are  recogni^d  by  their  concentric  striations  and  the  fact 
that  they  stain  blue  with  iodin  solutions  when  undi- 
gested, and  reddish,  due  to  erythrodextrin,  when  partially 
digested. 

Pathologically,  remnants  of  food  from  previous  meals, 
red  blood-corpuscles,  pus-cells,  sarcinae,  and  excessive 
numbers  of  yeast-cells  and  bacteria  may  be  encountered 
(Fig.  io6). 

Remnants  of  food  from  previous  meals  indicate 
deficient  gastric  motility. 

Red  Blood-corpuscles. — Blood  is  best  recognized  by 
the  chemic  tests  already  given.  The  corpuscles  some- 
times retain  a  fairly  normal  appearance,  but  are  generally 
so  degenerated  that  only  granular  pigment  is  left.  When 
only  a  few  fresh  looking  corpuscles  are  present,  they 
usually  come  from  irritation  of  the  mucous  membrane 
by  the  tube. 

Pus-cells. — Pus  is  rarely  encountered  in  the  fluid 
removed  after  a  test-meal.  Considerable  numbers  of 
pus-corpuscles  have  been  found  in  some  cases  of  gastric 
cancer.     Swallowed  sputum  must  always  be  considered. 

Sarcinae. — These  are  small  spheres  arranged  in  cuboid 
groups,  often  compared  to  bales  of  cotton.  They  fre- 
quently form  large  clumps  and  are  easily  recognized. 
They  stain  brown  with  iodin  solution.  They  signify  fer- 
mentation. Their  presence  is  strong  evidence  against 
the  existence  of  gastric  cancer,  in  which  disease  they 
rarely  occur. 

Yeast-cells. — As  already  stated,  a  few  yeast-cells 
may  be  found  under  normal  conditions.  The  presence 
of  considerable  numbers  is  e\adence  of  fermentation. 


EXAMINATION   OF  THE   GASTRIC  CONTENTS         303 

Their  appearance  has  been  described  (p.  171).     They 
stain  brown  with  iodin  solution. 

Bacteria. — Numerous  bacteria  may  be  encountered, 
especially  in  the  absence  of  free  hydrochloric  acid.  The 
Boas-Oppler  bacillus  is  the  only  one  of  special  significance. 
It  occurs  in  the  majority  of  cases  of  cancer,  and  is  rarely 
found  in  other  conditions.  Carcinoma  probably  fur- 
nishes a  favorable  medium  for  its  growth. 


Fig.  107. — Boas-Oppler  bacillus  from  case  of  gastric  cancer  (Boston). 

These  bacilli  (Fig.  107)  are  large  (5  to  10  [i  long), 
non-motile,  and  usually  arranged  in  clumps  or  end  to 
end  in  zig-zag  chains.  They  stain  brown  with  iodin 
solution,  which  distinguishes  them  from  Leptothrix  buc- 
calis  (p.  377),  which  is  not  infrequently  found  in  stomach 
fluid.  They  also  stain  by  Gram's  method.  They  are 
easily  seen  with  the  4  mm.  objective  in  unstained  prepa^ 
rations,  but  are  best  recognized  with  the  oil  lens,  after 
drying  some  of  the  fluid  upon  a  cover-glass,  fixing,  and 
staining  with  a  simple  bacterial  stain  or  by  Gram's 
method. 


304  THE  STOMACH 

A  few  large  non-motile  bacilli  are  frequently  seen ;  they 
cannot  be  called  Boas-Oppler  bacilli  unless  they  are 
numerous  and  show  something  of  the  typical  arrange- 
ment. 

E.    The  Gastric  Contents  in  Disease 

In  the  diagnosis  of  stomach  disorders  the  practitioner 
must  be  cautioned  against  relying  too  much  upon  exam- 
inations of  the  stomach-contents.  A  first  examination 
is  especially  unreliable.  Even  when  repeated  examina- 
tions are  made,  the  laboratory  findings  must  never  be 
considered  apart  from  the  clinical  signs. 

The  more  characteristic  findings  in  certain  disorders 
are  suggested  here. 

1 .  Dilatation  of  the  Stomach. — Evidences  of  retention 
and  fermentation  are  the  chief  characteristics  of  this 
condition.  Hydrochloric  acid  is  commonly  diminished. 
Pepsin  may  be  normal  or  sfightly  diminished.  Lactic 
acid  may  be  detected  in  small  amounts,  but  is  usually 
absent  when  the  stomach  has  been  washed  before  giving 
the  test-meal.  Both  motility  and  absorptive  power  are 
deficient.  The  microscope  commonly  shows  sarcinae, 
bacteria,  and  great  numbers  of  yeast-cells.  Remnants 
of  food  from  previous  meals  can  be  detected  with  the 
naked  eye  or  microscopically. 

2.  Gastric  Neuroses. — The  findings  are  variable. 
Successive  examinations  may  show  normal,  increased, 
or  diminished  hydrochloric  acid,  or  even  entire  absence 
of  the  free  acid.     Pepsin  is  usually  normal. 

In  the  neurosis  characterized  by  continuous  hyperse- 
cretion (gastrosuccorrhea) ,  40  c.c.  or  more  of  gastric  juice 
can  be   obtained   from   the   fasting  stomach.     Should 


EXAMINATION   OF  THE  GASTRIC  CONTENTS         305 

the  fluid  contain  food-particles,  it  is  probably  the  result 
of  retention,  not  hypersecretion. 

3.  Chronic  Gastritis. — Free  hydrochloric  acid  may  be 
increased  in  early  cases.  It  is  generally  diminished  in 
well-marked  cases,  and  is  often  absent  in  advanced 
cases.  Lactic  acid  is  often  present  in  traces,  rarely 
in  notable  amount.  Secretion  of  pepsin  and  rennin  is 
always  diminished  in  marked  cases.  Mucus  is  frequently 
present,  and  is  very  significant  of  the  disease.  Motility 
and  absorption  are  generally  deficient.  Small  fragments 
of  mucous  membrane  may  be  found,  and  when  examined 
by  a  pathologist,  may  occasionally  establish  the  diagnosis. 

4.  Achylia  Qastrica  (Atrophic  Gastritis). — This  con- 
dition may  be  a  terminal  stage  of  chronic  gastritis.  It  is 
sometimes  associated  with  the  blood-picture  of  pernicious 
anemia.  It  gives  a  great  decrease,  and  sometimes  entire 
absence  of  hydrochloric  acid  and  ferments.  The  total 
acidity  may  be  as  low  as  i  or  2  degrees.  Small  amounts 
of  lactic  acid  may  be  present.  Absorption  and  motihty 
are  usually  not  affected  greatly. 

5.  Gastric  Carcinoma. — As  far  as  the  laboratory 
examination  goes,  the  cardinal  signs  of  this  disease  are 
absence  of  free  hydrochloric  acid  and  presence  of  lactic 
acid  and  of  the  Boas-Oppler  bacillus.  These  findings 
are,  however,  by  no  means  constant. 

It  is  probable  that  some  substance  is  produced  by  the 
cancer  which  neutralizes  the  free  hydrochloric  acid,  and 
thus  causes  it  to  disappear  earlier  than  in  other  organic 
diseases  of  the  stomach. 

The  presence  of  lactic  acid  is  the  most  suggestive  single 
symptom  of  gastric  cancer.  In  the  great  majority  of 
cases  its  presence  in  notable  amount  (o.i  per  cent,  by 
20 


3o6  THE   STOMACH 

Strauss'  method)  after  Boas'  breakfast,  the  stomach 
having  been  washed  the  evening  before,  warrants  a 
tentative  diagnosis  of  mahgnancy. 

Carcinoma  seems  to  furnish  an  especially  favorable 
medium  for  the  growth  of  the  Boas-Oppler  bacillus,  hence 
this  micro-organism  is  frequently  present. 

Blood  can  be  detected  in  the  stomach  fluid  by  the 
chemic  tests  in  nearly  one-half  of  the  cases,  and  is  more 
common  when  the  new  growth  is  situated  at  the  pylorus. 
Blood  is  present  in  the  stool  in  nearly  every  case. 

Evidences  of  retention  and  fermentation  are  the  rule  in 
pyloric  cancer.  Tumor  particles  are  sometimes  found 
late  in  the  disease. 

6.  Gastric  Ulcer.— There  is  excess  of  free  hydrochloric 
acid  in  about  one-half  of  the  cases.  In  other  cases  the 
acid  is  normal  or  diminished.  Blood  is  often  present. 
The  diagnosis  must  be  based  largely  upon  the  clinical 
symptoms,  and  where  ulcer  is  strongly  suspected,  it  is 
probably  unwise  to  use  the  stomach-tube. 

IL  ADDITIONAL   EXAMINATIONS  WHICH   GIVE  INFOR- 
MATION AS  TO  THE  CONDITION  OF  THE  STOMACH 

1 .  Absorptive  Power  of  the  Stomach. — This  is  a  very 
unimportant  function,  only  a  few  substances  being  ab- 
sorbed in  the  stomach.  It  is  delayed  in  most  organic  dis- 
eases of  the  stomach,  especially  in  dilatation  and  carci- 
noma, but  not  in  neuroses.  The  test  has  Uttle  practical 
value. 

Give  the  patient,  upon  an  empty  stomach,  a  3-grain  cap- 
sule of  potassium  iodid  with  a  glass  of  water,  taking  care 
that  none  of  the  drug  adheres  to  the  outside  of  the  capsule. 


EXAinNATION  AS   TO  THE   CONDITION  OF  STOMACH    307 

At  intervals  test  the  saliva  for  iodids  by  moistening  starch- 
paper  with  it  and  touching  with  yellow  nitric  acid.  A  blue 
color  shows  the  presence  of  an  iodid,  and  appears  normally 
in  ten  to  fifteen  minutes  after  ingestion  of  the  capsule.  A 
longer  time  denotes  delayed  absorption. 

Starch  paper  is  prepared  by  soaking  filter-paper  in  boiled 
starch  and  drying. 

2.  Motor  Power  of  the  Stomach. — This  refers  to  the 
rapidity  with  which  the  stomach  passes  its  contents  on 
into  the  intestine^.  It  is  very  important :  intestinal  diges- 
tion can  compensate  for  insufficient  or  absent  stomach 
digestion  only  so  long  as  the  motor  power  is  good. 

Motility  is  impaired  to  some  extent  in  chronic  gastritis. 
It  is  especially  deficient  in  dilatation  of  the  stomach  due 
to  atony  of  the  gastric  wall  or  to  pyloric  obstruction, 
either  benign  or  maUgnant.  It  is  increased  in  most  con- 
ditions with  h^perchlorhydria. 

The  best  evidence  of  deficient  motor  power  is  the 
detection  of  food  in  the  stomach  at  a  time  when  it  should 
be  empty,  e.  g.,  before  breakfast  in  the  morning.  WTien 
more  than  60  c.c.  of  fluid  are  obtained  with  the  tube  one 
hour  after  a  Ewald  breakfast,  deficient  motility  may  be 
inferred. 

Ewald's  salol  test  is  scarcely  so  reliable  as  the  above.  It 
depends  upon  the  fact  that  salol  is  not  absorbed  until  it 
reaches  the  intestines  and  is  decomposed  by  the  alkaline 
intestinal  juices. 

The  patient  is  given  15  grains  of  salol  with  a  test-breakfast, 
and  the  urine,  passed  at  intervals  thereafter,  is  tested  for 
salicyluric  acid.  A  few  drops  of  10  per  cent,  ferric  chlorid 
solution  are  added  to  a  small  quantity  of  the  urine.    A  violet 


3o8  THE   STOMACH 

color  denotes  the  presence  of  salicyluric  acid.  It  appears 
normally  in  sixty  to  seventy- live  minutes  after  ingestion  of  the 
salol.     A  longer  time  indicates  impaired  motor  power. 

3.  To  Determine  Size  and  Position  of  Stomach. — 

After  removing  the  test-meal,  while  the  tube  is  still  in 
place,  force  quick  puffs  of  air  into  the  stomach  by  com- 
pression of  the  bulb.  The  puflfs  can  be  clearly  heard  with 
a  stethoscope  over  the  region  of  the  stomach,  and  no- 
where else. 

If  desired,  the  patient  may  be  given  a  dram  of  sodium 
bicarbonate  in  solution,  followed  immediately  by  the 
same  amount  of  tartaric  acid,  also  in  solution;  or  he  may 
take  the  two  parts  of  a  seidlitz  powder  separately.  The 
carbon  dioxid  evolved  distends  the  stomach,  and  its 
outline  can  easily  be  determined  by  percussion. 

4.  Sahli's  Desmoid  Test  of  Gastric  Digestion. — Two 
pills,  one  containing  o.i  gram  iodoform,  the  other  0.05 
gram  methylene-blue,  are  wrapped  in  little  bags  made  of 
thin  sheets  of  rubber  and  tied  with  a  string  of  catgut. 
The  bags  must  be  carefully  folded  and  tied.  For  detailed 
directions  the  reader  is  referred  to  Sahli's  book,  Diag- 
nostic Methods. 

The  patient  swallows  the  two  bags  with  the  aid  of  a 
little  water  during  the  noon  meal,  and  the  urine  is  tested 
at  intervals  thereafter.  According  to  Sahli,  the  catgut 
is  digested  by  gastric  juice  and  not  by  pancreatic  or 
intestinal  juices.  If  gastric  digestion  is  normal,  iodin 
and  methylene-blue  can  be  detected  in  the  urine  in  the 
afternoon  or  evening  of  the  same  day.  The  reaction 
may  occur  when  digestion  is  very  poor,  provided  gastric 
motility  is  diminished,  but  it  is  then  delayed.     If  the 


EXAMINATION  AS   TO  THE   CONDITION   OP  STOMACH   309 

reaction   does    not.  appear,   gastric   digestion   has   not 
occurred. 

Methylene-blue  is  recognized  in  the  urine  by  the  green  or 
blue  color  which  it  imparts.  It  is  sometimes  eliminated  as 
a  chromogen;  and  a  little  of  the  urine  must  be  acidified  with 
acetic  acid  and  boiled  to  bring  out  the  color. 

To  detect  the  iodin,  some  of  the  urine  is  decolorized  by 
gently  heating  and  filtering  through  animal  charcoal.  To 
10  c.c.  are  then  added  i  c.c.  dilute  sulphuric  acid,  and  0.5 
c.c.  of  a  I  per  cent,  solution  of  sodium  nitrite  and  2  c.c.  of 
chloroform.  Upon  shaking,  a  rose  color  will  be  imparted  to 
the  chloroform  if  iodin  be  present. 


CHAPTER  V 
THE  FECES 

As  commonly  practised,  an  examination  of  the  feces  is 
limited  to  a  search  for  intestinal  parasites  or  their  ova. 
Much  of  value  can,  however,  be  learned  from  other  simple 
examinations,  particularly  a  careful  inspection.  Anything 
approaching  a  complete  analysis  is,  on  the'other  hand,  a 
waste  of  time  for  the  clinician. 

The  normal  stool  is  a  mixture  of — (a)  water;  (b) 
undigested  and  indigestible  remnants  of  food,  as  starch- 
granules,  particles  of  meat,  plant-cells  and  fibers,  etc.; 
(c)  digested  foods,  carried  out  before  absorption  could 
take  place;  (d)  products  of  the  digestive  tract,  as  altered 
bile-pigments,  mucus,  etc. ;  (e)  products  of  decomposition, 
as  indol,  skatol,  fatty  acids,  and  various  gases;  (/)  epi- 
thelial cells  shed  from  the  wall  of  the  intestinal  canal; 
(g)  harmless  bacteria,  which  are  always  present  in 
enormous  numbers. 

Pathologically,  we  may  find  abnormal  amounts  of 
normal  constituents,  blood,  pathogenic  bacteria,  animal 
parasites  and  their  ova,  and  biliary  and  intestinal  con- 
cretions. 

The  stool  to  be  examined  should  be  passed  into  a  clean 
vessel,  without  admixture  of  urine.  The  offensive  odor 
can  be  partially  overcome  with  turpentine  or  5  per  cent, 
phenol.  When  search  for  amebae  is  to  be  made,  the 
vessel  must  be  warm,  and  the  stool  kept  warm  until 
examined;  naturally,  no  disinfectant  can  be  used. 

310 


MACROSCOPIC  EXAMINATION  3 II 

I.  MACROSCOPIC  EXAMINATION 

1.  Quantity. — The  amount  varies  greatly  with  diet 
and  other  factors.  The  average  is  about  loo  to  150  gm. 
in  twenty-four  hours. 

2.  Frequency. — One  or  two  stools  in  twenty-four  hours 
may  be  considered  normal,  yet  one  in  three  or  four  days 
is  not  uncommon  with  healthy  persons.  The  individual 
habit  should  be  considered  in  every  case. 

3.  Form  and  Consistence. — Soft,  mushy,  or  liquid 
stools  follow  cathartics  and  accompany  diarrhea.  Co- 
pious, purely  serous  discharges  without  fecal  matter 
are  significant  of  Asiatic  cholera,  although  sometimes 
observed  in  other  conditions.  Hard  stools  accompany 
constipation.  Rounded  scybalous  masses  are  common  in 
habitual  constipation,  and  indicate  atony  of  the  muscular 
coat  of  the  intestines  Flattened,  ribbon-like  stools  re- 
sult from  some  obstruction  in  the  rectum,  generally  a 
tumor  or  stricture  from  a  healed  ulcer,  most  commonly 
syphilitic.  When  bleeding  piles  are  absent,  blood- 
streaks  upon  such  a  stool  point  to  carcinoma. 

4.  Color. — The  normal  light  or  dark-brown  color  is  due 
chiefly  to  hydrobilirubin,  which  is  formed  from  biHrubin 
by  reducing  processes  in  the  intestines,  largely  the  result 
of  bacterial  activity.  The  stools  of  infants  are  yellow, 
owing  partly  to  their  milk  diet  and  partly  to  the  presence 
of  unchanged  bihrubin. 

Diet  and  drugs  cause  marked  changes:  milk,  a  light 
yellow  color;  cocoa  and  chocolate,  dark  gray;  various 
fruits,  reddish  or  black ;  iron  and  bismuth,  dark  brown  or 
black;  hematoxylin,  red,  etc. 

Pathologically,  the  color  is  important.  A  golden  yellow 


312  THE  FECES 

is  generally  due  to  unchanged  bilirubin.  Green  stools 
are  not  uncommon,  especially  in  diarrheas  of  childhood. 
The  color  is  due  to  biliverdin  or,  sometimes,  to  chromo- 
genic  bacteria.  Putty-colored  or  "  acholic  "  stools  occur 
when  bile  is  deficient,  either  from  obstruction  to  outflow 
or  from  deficient  secretion.  The  color  is  due  less  to 
absence  of  bile-pigments  than  to  presence  of  fat.  Similar 
stools  are  common  in  conditions  Hke  tuberculous  peri- 
tonitis, which  interfere  with  absorption  of  fats,  and  in 
pancreatic  disease. 

Notable  amounts  of  blood  produce  tarry  black  stools 
when  the  source  of  the  hemorrhage  is  the  stomach  or 
upper  intestine,  and  a  dark  brown  or  bright  red  as  the 
source  is  nearer  the  rectum.  When  diarrhea  exists  the 
color  may  be  red,  even  if  the  source  of  the  blood  is  high 
up.  Red  streaks  of  blood  upon  the  outside  of  the  stool 
are  due  to  lesions  of  rectum  or  anus. 

5.  Odor. — Products  of  decomposition,  chiefly  indol 
and  skatol,  are  responsible  for  the  normal  ofifensive  odor. 
A  sour  odor  is  normal  for  nursing  infants,  and  is  noted  in 
mild  diarrheas  of  older  children.  In  the  severe  diarrheas 
of  childhood  a  putrid  odor  is  common.  In  adults,  stools 
emitting  a  very  foul  stench  are  suggestive  of  malignant 
or  s}T3hiUtic  ulceration  of  the  rectum  or  gangrenous 
dysentery. 

6.  Mucus. — Excessive  quantities  of  mucus  are  easily 
detected  with  the  naked  eye,  and  signify  irritation  or 
inflammation.  When  the  mucus  is  small  in  amount  and 
intimately  mixed  with  the  stool,  the  trouble  is  probably 
in  the  small  intestine.  Larger  amounts,  not  well  mixed 
with  fecal  matter,  indicate  inflammation  of  the  large 
intestine.     Stools  composed  almost  wholly  of  mucus  and 


MACROSCOPIC  EXAMINATION  313 

streaked  with  blood  are  the  rule  in  dysentery,  ileocolitis, 
and  intussusception. 

In  the  so-called  mucous  colic  or  membranous  enteritis 
shreds  and  ribbons  of  altered  mucus,  sometimes  represent- 
ing complete  casts  of  portions  of  the  bowel,  are  passed. 
The  mucus  sometimes  takes  the  form  of  frog-spawn-like 
masses.  In  some  cases  it  is  passed  at  variable  intervals, 
with  cohc;  in  others,  with  every  stool,  with  only  vague 
pains  and  discomfort.  It  is  distinguished  from  inflam- 
matory mucus  by  absence  of  pus-corpuscles.  The  con- 
dition is  not  uncommon  and  should  be  more  frequently 
recognized.  It  is  probably  a  secretory  neurosis,  hence 
the  name  "membranous  enteritis"  is  inappropriate. 

7.  Concretions. — Gall-stones  are  probably  more  com- 
mon than  is  generally  supposed,  and  should  be  searched 
for  in  every  case  of  obscure  colicky  abdominal  pain. 
Intestinal  concretions  (enteroliths)  are  rare.  Intestinal 
sand,  consisting  of  sand-like  grains,  is  especially  common 
in  neurotic  conditions,  such  as  mucous  colitis. 

Concretions  can  be  found  by  breaking  up  the  fecal 
matter  in  a  sieve  (which  may  be  improvised  from  gauze) 
while  pouring  water  over  it.  It  must  be  remembered  that 
gall-stones,  if  soft,  may  go  to  pieces  in  the  bowel. 

8.  Animal  Parasites. — Segments  of  tapeworms  and 
the  adults  and  larvae  of  other  parasites  are  often  found  in 
the  stool.  They  are  best  searched  for  in  the  manner 
described  for  concretions.  The  search  should  be  pre- 
ceded by  a  vermicide  and  a  brisk  purge.  Patients  fre- 
quently mistake  vegetable  tissue  (long  fibers  from  poorly 
masticated  celery  or  "  greens,"  cells  from  oranges,  etc.) 
for  intestinal  parasites,  and  the  writer  has  known 
physicians  to  make  similar  mistakes.     Even  slight  famil- 


314  THE  FECES 

iarity  with  the  microscopic  structure  of  vegetable  tissue 
will  prevent  the  chagrin  of  such  errors. 

9.  Curds. — The  stools  of  nursing  infants  frequently 
contain  whitish  curd-like  masses,  due  either  to  imperfect 
digestion  of  fat  or  casein  or  to  excess  of  these  in  the  diet. 
When  composed  of  fat,  the  masses  are  soluble  in  ether, 
and  give  the  Sudan  III  test.  If  composed  of  casein, 
they  will  become  tough  and  fibrous-like  when  placed  in 
formalin  (10  per  cent.)  for  twenty-four  hours. 

11.  CHEMIC  EXAMINATION 

Complicated  chemic  examinations  are  of  little  value  to 
the  clinician.     Certain  tests  are,  however,  important. 

1.  Blood. — When  present  in  large  amount  blood  pro- 
duces such  changes  in  the  appearance  of  the  stool  that  it 
is  not  likely  to  be  overlooked.  Traces  of  blood  (occult 
hemorrhage)  can  be  detected  only  by  special  tests. 
Recognition  of  occult  hemorrhage  has  its  greatest  value 
in  diagnosis  of  gastric  cancer  and  ulcer.  It  is  constantly 
present  in  practically  every  case  of  gastric  cancer,  and  is 
always  present,  although  usually  intermittently,  in  ulcer. 
Traces  of  blood  also  accompany  malignant  disease  of  the 
bowel,  the  presence  of  certain  intestinal  parasites,  and 
other  conditions. 

Detection  of  Occult  Hemorrhage. — Soften  a  portion  of  the 
stool  with  water,  shake  with  an  equal  volume  of  ether  to 
remove  fat,  and  discard  the  ether.  Treat  the  remaining 
material  with  about  one-third  its  volume  of  glacial  acetic 
acid  and  extract  with  ether.  Should  the  ether  not  separate 
well,  add  a  little  alcohol.  Apply  the  guaiac  test  to  the  ether 
as  already  described  (p.  125). 


MICROSCOPIC  EXAMINATION  315 

In  every  case  iron-containing  medicines  must  be  stopped, 
and  blood-pigment  must  be  excluded  from  the  food  by  giving 
an  appropriate  diet,  e.  g.,  bread,  milk,  eggs,  and  fruit.  At  the 
beginning  of  the  restricted  diet  give  a  dram  of  powdered 
charcoal,  or  7  grains  of  carmin,  so  as  to  mark  the  correspond- 
ing stool. 

2.  Bile. — Normally,  unaltered  bile-pigment  is  never 
present  in  the  feces  of  adults.  In  catarrhal  conditions 
of  the  small  intestine  bilirubin  may  be  carried  through  un- 
changed. It  may  be  demonstrated  by  filtering  (after 
mixing  with  water  if  the  stool  be  solid)  and  testing  the 
filtrate  by  Gmelin's  method,  as  described  under  The 
Urine. 

Hydrobilirubin  will  give  a  red  color  if  a  little  of  the 
stool  be  rubbed  up  with  saturated  mercuric  chlorid 
solution  and  allowed  to  stand  twenty-four  hours.  The 
red  color  is  likewise  imparted  to  microscopic  structures 
which  are  stained  with  hydrobilirubin.  A  green  color 
in  this  test  shows  the  presence  of  unchanged  bilirubin. 

III.  MICROSCOPIC  EXAMINATION 

Care  must  be  exercised  in  selection  of  portions  for 
examination.  A  random  search  will  often  reveal  nothing 
of  interest.  A  small  bit  of  the  stool,  or  any  suspicious- 
looking  particle,  is  placed  upon  a  slide,  softened  with 
water  if  necessary,  and  pressed  out  into  a  thin  layer  with 
a  cover-glass.  A  large  slide — about  2  by  3  inches — 
with  a  correspondingly  large  cover  will  be  found  conve- 
nient. Most  of  the  structures  which  it  is  desired  to  see 
can  be  found  with  a  16  mm.  objective.  Details  of  struc- 
ture must  be  studied  with  a  higher  power. 


3i6 


THE   FECES 


The  bulk  of  the  stool  consists  of  granular  debris. 
Among  the  recognizable  structures  met  in  normal  and 
pathologic  conditions  are:  Remnants  of  food,  epithelial 
cells,  pus-corpuscles,  red  blood-corpuscles,  crystals,  bac- 
teria, and  ova  of  animal  parasites  (Fig.  io8). 

I .  Remnants  of  Food. — These  include  a  great  variety 
of  structures  which  are  very  confusing  to  the  student. 
Considerable  study  of  normal  feces  is  necessary  for  their 
recognition. 


mm 


Fig.  io8. — Microscopic  elements  of  normal  feces:  a.  Muscle-fibers;  b,  connective 
tissue;  c,  epithelial  cells;  d,  white  blood -corpuscles;  e.  spiral  vessels  of  plants;  f-h,  vege- 
table cells;  J,  plant  hairs;  k,  triple  phosphate  crystals;  /,  stone  cells.  Scattered  among 
these  elements  are  micro-organisms  and  debris  (after  v.  Jaksch). 


Vegetable  fibers  are  generalh'  recognized  from  their 
spiral  structure  or  their  pits,  dots,  or  reticulate  mark- 
ings; vegetable  cells,  from  their  double  contour  and  the 
chlorophyl  bodies  which  many  of  them  contain.  These 
cells  are  apt  to  be  mistaken  for  the  ova  of  parasites. 
Starch-granules  sometimes  retain  their  original  form,  but 
are  ordinarily  not  to  be  recognized  except  by  their  stain- 
ing reaction.  They  strike  a  blue  color  with  Lugol's  solu- 


MICROSCOPIC  EXAMINATION 


317 


tion  when  undigested;  a  red  color,  when  slightly  digested. 
Muscle-fibers  are  yellow,  and  when  poorly  digested  appear 
as  short,  transversely  striated  cylinders  with  rather 
squarely  broken  ends  (Fig.  109).  Generally,  the  ends 
are  rounded  and  the  striations  faint,  or  only  irregularly 
round  or  oval  yellow  masses  are  found.  Curds  of  milk 
are  especially  important  in  the  stools  of  children.  They 
must  be  distinguished  from  small  masses  of  Jat  (p.  314). 


Fig.  log.- 


-Poorly  digested  muscle-fiber  in  feces  showing  striations  (X  200)  (photograph 
by  the  author). 


Excess  of  any  of  these  structures  may  result  from 
excessive  ingestion  or  deficient  intestinal  digestion. 

2.  Epithelial  Cells. — A  few  cells  derived  from  the 
wall  of  the  alimentary  canal  are  a  constant  finding.  They 
show  all  stages  of  degeneration,  and  are  often  unrecog- 
nizable. A  marked  excess  has  its  origin  in  a  catarrhal 
condition  of  some  part  of  the  bowel.  Squamous  cells 
come  from  the  anal  orifice;  otherwise  the  form  of  the 
cells  gives  no  clue  to  the  location  of  the  lesion. 


3l8  THE   FECES 

3.  Pus. — Amounts  of  pus  sufficient  to  be  recognized 
with  the  eye  alone  indicate  rupture  of  an  abscess  into 
the  bowel.  If  well  mixed  with  the  stool,  the  source  is 
high  up,  but  in  such  cases  the  pus  is  apt  to  be  more  or 
less  completely  digested,  and  hence  unrecognizable. 
Small  amounts,  detected  only  by  the  microscope,  are 
present  in  catarrhal  and  ulcerative  conditions  of  the  in- 
testine, the  number  of  pus-cells  corresponding  to  the 
severity  and  extent  of  the  process. 

4.  Blood=corpuscles. — Unaltered  red  corpuscles  are 
rarely  found  unless  their  source  is  near  the  anus.  Ordi- 
narily, only  masses  of  blood-pigment  can  be  seen.  Blood 
is  best  recognized  by  the  chemic  tests  fp.  274). 

5.  Bacteria. — In  health,  bacteria  constitute  about  one- 
third  of  the  weight  of  the  dried  stool.  They  are  beneficial 
to  the  organism,  although  not  actually  necessary  to  its 
existence.  It  is  both  difficult  and  unprofitable  to  iden- 
tify them.  The  great  majority  belong  to  the  colon 
bacillus  group,  and  are  negative  to  Gram's  method  of 
staining. 

In  some  pathologic  conditions  the  character  of  the 
intestinal  flora  changes,  so  that  Gram-staining  bacteria 
very  greatly  predominate.  As  shown  by  R.  Schmidt, 
of  Neusser's  clinic  in  Vienna,  this  change  is  most  constant 
and  most  striking  in  cancer  of  the  stomach,  owing  to 
large  numbers  of  Boas-Oppler  bacilli,  and  is  of  consider- 
able value  in  diagnosis.  He  believes  that  a  diagnosis 
of  gastric  carcinoma  should  be  very  unwillingly  made 
with  an  exclusively  "Gram-negative"  stool,  while  a 
"Gram-positive"  stool,  due  to  bacilli  (which  should  also 
stain  brown  with  Lugol's  solution),  may  be  taken  as  very 
strong  evidence  of  cancer.     A  Gram-positive  stool  due 


MICROSCOPIC  EXAMINATION  319 

to  cocci  is  suggestive  of  intestinal  ulceration.  The 
technic  is  the  same  as  when  Gram's  method  is  applied 
to  other  material  (p.  409),  except  that  the  smear  is  fixed 
by  immersion  in  methyl-alcohol  for  five  minutes  instead 
of  by  heat.  Fuchsin  is  the  best  counterstain.  The  deep 
purple  Gram-staining  bacteria  stand  out  much  more 
prominently  than  the  pale-red  Gram-negative  organisms, 
and  one  may  be  misled  into  thinking  them  more  numer- 
ous even  in  cases  in  which  they  are  much  in  the  minority. 
The  number  of  Boas-Oppler  bacilli  can  be  increased  by 
administering  a  few  ounces  of  sugar  of  milk  the  day 
before  the  examination.  The  bacteria  can  be  obtained 
comparatively  free  from  food  remnants  by  mixing  a 
little  of  the  feces  with  water,  allowing  to  settle  for  a 
short  time,  and  making  smears  from  the  supernatant 
fluid. 

Owing  to  the  difl&culty  of  excluding  swallowed  sputum, 
the  presence  of  the  tubercle  bacillus  is  less  significant  in 
the  feces  than  in  other  material.  It  may,  however,  be 
taken  as  evidence  of  intestinal  tuberculosis  when  clinical 
signs  indicate  an  intestinal  lesion  and  reasonable  care  is 
exercised  in  regard  to  the  sputum.  Success  in  the 
search  will  depend  largely  upon  careful  selection  of  the 
portion  examined.  A  random  search  will  almost  surely 
fail.  Whitish  or  grayish  flakes  of  mucus  or  blood- 
stained or  purulent  particles  should  be  spread  upon 
slides  or  covers  and  stained  by  the  method  given  upon  p. 
168.  In  the  case  of  rectal  ulcers,  swabs  can  be  made 
directly  from  the  ulcerated  surface. 

6.  Crystals. — Various  crystals  may  be  found,  but  few 
have  any  significance.  Slender,  needle-like  crystals  of 
fatty  acids  and  soaps  (Fig.  36)  and  triple  phosphate 


320  THE  FECES 

crystals  (Fig.  io8)  are  common.  Characteristic  octahe- 
dral crystals  of  calcium  oxalate  (Fig.  51)  appear  after  in- 
gestion of  certain  vegetables.  Charcot-Leyden  crystals 
(Fig.  9)  are  not  infrequently  encountered,  and  strongly 
suggest  the  presence  of  intestinal  parasites.  Yellowish 
or  brown,  needle-like  or  rhombic  crystals  of  hematoidin 
(Fig.  36)  may  be  seen  after  hemorrhage  into  the  bowel. 
7.  Parasites  and  Ova. — The  stool  should  be  well  mixed 
with  water  and  allowed  to  settle.  The  ova  will  be  found 
in  the  upper  or  middle  portions  of  the  sediment.  The 
flagellates  are  best  found  in  the  liquid  stool  after  a  dose 
of  salts.  Descriptions  will  be  found  in  the  following 
chapter. 

IV.  FUNCTIONAL  TESTS 

1.  Schmidt's  Test  Diet. — Much  can  be  learned  of  the 
various  digestive  functions  from  a  microscopic  study  of 
the  feces,  especially  when  the  patient  is  upon  a  known 
diet.  For  this  purpose  the  standard  diet  of  Schmidt  is 
generally  adopted.     This  consists  of: 

Morning 0.5  liter  milk  and  50  gm.  toast. 

Forenoon 0.5  liter  porridge,  made  as  follows:  40  gm. 

oatmeal,  10  gm.  butter,  200  c.c.  milk, 
300  c.c.  water,  and  one  egg. 

Midday 125  gm.  hashed  meat,  with  20  gm.  butter, 

fried  so  that  the  interior  is  quite  rare; 
250  gm.  potato,  made  by  cooking  190 
gm.  potato  with  100  c.c.  milk  and  10  gm. 
butter,  the  whole  boiled  down  to  250  c.c. 

Afternoon Same  as  morning. 

Evening Same  as  forenoon. 

At  the  beginning  of  the  diet,  the  stool  should  be 
marked  off  with  carmin  or  charcoal.     One  should  famil- 


FUNCTIONAL   TESTS  32 1 

iarize  himself  with  the  microscopic  appearance  of  the 
feces  of  normal  persons  upon  this  diet. 

Deficiency  of  starch  digestion  is  recognized  by  the 
number  of  starch-granules  which  strike  a  blue  color  with 
iodin.  With  exception  of  those  inclosed  in  plant  cells 
none  are  present  normally. 

The  degree  of  protein  digestion  is  ascertained  by  the 
appearance  of  the  muscle-fibers.  Striations  are  clearly 
visible  only  when  digestion  is  imperfect  (Fig.  109).  Ac- 
cording to  Schmidt,  the  presence  of  nuclei  in  muscle-fibers 
denotes  complete  absence  of  pancreatic  function.  The 
presence  of  connective-tissue  shreds  indicates  deficient 
gastric  digestion,  since  raw  connective  tissue  is  digested 
only  in  the  stomach.  These  shreds  can  be  recognized 
macroscopically  by  examining  in  a  thin  layer  against  a 
black  background,  and  microscopically  by  their  fibrous 
structure  and  the  fact  that  they  clear  up  when  treated 
with  acetic  acid. 

Digestion  of  fats  is  checked  up  by  the  amount  of 
neutral  fat. 

2.  Sahli's  Qlutoid  Test, — The  Schmidt  test  diet  in- 
volves some  inconvenience  for  the  patient,  and  inter- 
pretation of  results  requires  much  experience  upon  the 
part  of  the  physician.  A  number  of  other  methods  of 
testing  the  digestive  functions  have  been  proposed.  The 
glutoid  test  of  SahH  is  one  of  the  most  satisfactory. 
This  is  similar  to  his  desmoid  test  of  gastric  digestion 
described  on  page  308.  A  glutoid  capsule  containing 
0.15  gram  iodoform  is  taken  with  an  Ewald  breakfast. 
The  capsule  is  not  digested  by  the  stomach  fluid,  but 
is  readily  digested  by  pancreatic  juice.  Appearance  of 
iodin  in  the  saUva  and  urine  within  four  to  six  hours 
21 


322  THE   FECES 

indicates  normal  gastric  motility,  normal  intestinal  di- 
gestion, and  normal  absorption.  Instead  of  iodoform, 
0.5  gram  salol  may  be  used,  salicyluric  acid  appearing 
in  the  urine  in  about  the  same  time.  For  tests  for  iodin 
and  salicyluric  acid,  see  pages  307  and  309. 

The  glutoid  capsules  are  prepared  by  soaking  gelatin 
capsules  in  formalin.  Sahli  states  that  filled  capsules 
can  be  purchased  of  A.  G.  Haussmann,  in  St.  Gall, 
Switzerland. 

3.  Miiller's  Test  for  Trypsin. — A  calomel  purge  is 
given  two  hours  after  a  meal.  Particles  of  the  feces  are 
placed  upon  solidified  blood-serum.  This  is  incubated 
at  a  temperature  of  55°  to  60°  C.  to  prevent  action  of 
bacteria.  Digestion  of  the  serum — indicated  by  a 
translucent,  roughened,  depressed  surface — presumably 
shows  the  presence  of  trypsin,  and  indicates  pancreatic 
sufficiency.  Trypsin  can  seldom  be  detected  without 
the  preceding  purge. 


CHAPTER  VI 
ANIMAL  PARASITES 

Animal  parasites  are  common  in  all  countries,  but  are 
especially  abundant  in  the  tropics,  where,  in  some  places, 
almost  every  native  is  host  for  one  or  more  species. 
Because  of  our  growing  intercourse  with  these  regions 
the  subject  is  assuming  increasing  importance  in  this 
country.  Many  parasites,  hitherto  comparatively  un- 
known here,  will  probably  become  more  common. 

Some  parasites  produce  no  symptoms,  even  when 
present  in  large  numbers.  Others  cause  very  serious 
symptoms.  It  is,  however,  impossible  to  make  a  sharp 
distinction  between  pathogenic  and  non-pathogenic 
varieties.  Parasites  which  cause  no  apparent  ill  effects 
in  one  individual  may,  under  certain  conditions,  produce 
marked  disturbances  in  another.  The  disturbances  are 
so  varied,  and  frequently  so  indefinite,  that  diagnosis  can 
rarely  be  made  from  the  clinical  symptoms.  It  must  rest 
upon  detection,  by  the  naked  eye  or  the  microscope,  of 
(a)  the  parasites  themselves,  (b)  their  ova  or  young 
progeny,  or  (c)  some  of  their  products. 

Unlike  bacteria,  the  great  majority  of  animal  parasites 
-multiply  by  means  of  alternating  and  differently  formed 
generations,  which  require  widely  different  conditions 
for  their  development.  The  few  exceptions  are  chiefly 
among  the  protozoa.     Multiplication  of  parasites  within 

323 


324  ANIMAL  PARASITES 

the  same  host  is  thus  prevented.  In  the  case  of  the 
hook-worm,  for  example,  there  is  no  increase  in  the  num- 
ber of  worms  in  the  host's  intestine,  except  through  re- 
infection from  the  outside.  The  young  are  carried  out 
of  the  intestine  and  must  pass  a  certain  period  of  devel- 
opment in  warm,  moist  earth  before  they  can  again 
enter  the  human  body  and  grow  to  maturity.  In  general, 
this  alternation  of  periods  of  development  takes  place  in 
one  of  three  ways: 

(i)  The  young  remain  within  the  original  host,  but 
travel  to  other  organs,  where  they  do  not  reach  maturity, 
but  lie  quiescent  until  taken  in  by  a  new  host.  A  good 
example  is  Trichinella  spiralis. 

(2)  The  young  or  the  ova  which  subsequently  hatch 
pass  out  of  the  host,  and  either  {a)  go  through  a  simple 
process  of  growth  and  development  before  entering 
another  host,  as  is  the  case  with  the  hook-worm,  or  {b) 
pass  through  one  or  more  free-living  generations,  the 
progeny  of  which  infect  new  hosts,  as  is  the  case  with 
Strongyloides  intestinalis. 

(3)  The  young  or  ova  or  certain  specialized  forms 
either  directly  {e.  g.,  malarial  parasites)  or  indirectly 
(e.  g.,  tapeworms)  reach  a  second  host  of  different 
species,  where  a  widely  different  process  of  development 
occurs.  The  host  in  which  the  adult  or  sexual  existence 
is  passed  is  called  the  'definitive  or  final  host;  that  in 
which  the  intermediate  or  larval  stage  occurs,  the 
intermediate  host.  Man,  for  example,  is  the  definitive 
host  for  TcEuia  saginata,  and  the  intermediate  host  for 
the  malarial  parasites  and  Tcenia  echinococcus. 

A  few  words  concerning  the  classification  and  nomen- 
clature of  living  organisms  in  general  will  be  helpful 


ANIMAL  PARASITES  325 

at  this  place.  Individuals  which  are  alike  in  all  essential 
respects  are  classed  together  as  a  species.  Closely  related 
species  are  grouped  together  to  form  a  genus;  genera 
which  have  certain  characteristics  in  common  make  up 
a  family;  families  are  grouped  into  orders;  orders  into 
classes;  and  classes,  finally,  into  the  branches  or  phyla, 
which  make  up  the  kingdom.  In  some  cases  these  groups 
are  subdivided  into  intermediate  groups — subphyla, 
subfamilies,  etc.,  and  occasionally,  slight  differences 
warrant  subdivision  of  the  species  into  varieties.  The 
animal  kingdom  comprises  nine  branches:  Protozoa, 
Porifera,  Ccelenterata,  Echinodermata,  Vermidea,  Arth- 
ropoda,  Mollusca,  Prochordata,  and  Chordata. 

The  scientific  name  of  an  animal  or  plant  consists  of 
two  parts,  both  Latin  or  Latinized  words,  and  is  printed 
in  italics.  The  first  part  is  the  name  of  the  genus  and 
begins  with  a  capital  letter;  the  second  is  the  name  of  the 
species  and  begins  with  a  lower  case  letter,  even  when  it 
was  originally  a  proper  name.  When  there  are  varieties 
of  a  species,  a  third  part,  the  designation  of  the  variety, 
is  appended.  The  author  of  the  name  is  sometimes  in- 
dicated in  Roman  type  immediately  after  the  name 
of  the  species.  Examples:  Spirochceta  vincenti,  often 
abbreviated  to  Sp.  vincenti  when  the  genus  name  has 
been  used  just  previously;  Staphylococcus  pyogenes 
albus;  Necator  americanus,  Stiles. 

At  the  present  time  there  is  great  confusion  in  the 
.naming  and  classification  of  parasites.  Some  have  been 
given  a  very  large  number  of  names  by  different  observers, 
and  in  many  cases  different  parasites  have  been  described 
under  the  same  name.  The  alternation  of  generations 
and  the  marked  differences  in  some  cases  between  male 


326  ANIMAL  PARASITES 

and  female  have  contributed  to  the  confusion,  different 
forms  of  the  same  parasite  being  described  as  totally 
unrelated  species. 

The  number  of  parasites  which  have  been  described 
as  occurring  in  man  and  the  animals  is  extremely  large. 
Only  those  which  are  of  medical  interest  are  mentioned 
here.  They  belong  to  three  phyla — Protozoa,  Vermidea, 
and  Arthropoda. 

PHYLUM  PROTOZOA 

These  are  unicellular  organisms,  the  simplest  types 
of  animal  life.  There  is  very  little  differentiation  of 
structure.  Each  contains  at  least  one,  and  some  several 
nuclei.  Some  contain  contractile  vacuoles;  some  have 
cilia  or  flagella  as  special  organs  of  locomotion.  They 
reproduce  by  division,  by  budding,  or  by  sporulation. 
Sometimes  there  is  an  alternation  of  generations,  in  one 
of  which  sexual  processes  appear,  as  is  the  case  with  the 
malarial  parasites.  The  protozoa  are  very  numerous, 
the  subphylum  Sarcodina  alone  including  no  less  than 
5000  species.  Most  of  the  protozoa  are  microscopic  in 
size ;  a  few  are  barely  visible  to  the  naked  eye.  One  can 
gain  a  general  idea  of  their  appearance  by  examining 
water  (together  with  a  little  of  the  sediment)  from  the 
bottom  of  any  pond.  Such  water  usually  contains  amebae 
and  a  considerable  variety  of  ciliated  and  flagellated 
forms. 

The  following  is  an  outline  of  those  protozoa  which 
are  of  medical  interest,  together  vvith  the  subphyla  and 
classes  to  which  they  belong. 


PHYLUM  PROTOZOA 


327 


PHYLUM  PROTOZOA 

SuBPHYLUM  I.     SARCODINA. — Locomotion   by  means  of  pseudo- 
podia. 

Class  Rhizopoda. — Pseudopodia  form  lobose  or  reticulose  processes. 


Genus. 
Entamoeba. 


Species. 
E.  histolytica. 
E.  tetragena. 
E.  coli. 
E.  buccalis. 


StJBPHYLUM  II.  MASTIGOPHORA  (FLAGELLATA).— Locomotion 
by  means  of  flagella. 

Class  Zoomastigophora. — Forms  in  which  animal  characteristics  pre- 
dominate. 


Genus. 

species. 

Spirochaeta. 

Sp.  obermeieri. 

Sp.  vincenti. 

Sp.  buccalis. 

Sp.  dentium. 

Sp.  refringens. 

Treponema. 

T.  pallidum. 

T.  pertenue. 

Trypanosoma. 

T.  gambiense. 

T.  cruzi. 

T.  lewisi. 

T.  evansi. 

T.  brucei. 

T.  equiperdum. 

Leishmania. 

L.  donovani. 

L.  tropica. 

L.  infantum. 

Cercomonas. 

C.  hominis. 

Bodo. 

B.  urinarius. 

Trichomonas. 

T.  vaginalis. 

T.  intestinalis. 

T.  pulmonalis. 

Lamblia. 

L.  intestinalis. 

328  ANIMAL  PARASITES 

SuBPHYLUM  III.     SPOROZOA.— All  members  parasitic.     Propaga- 
tion by  means  of  spores.     No  special  organs  of  locomotion. 
Class  Telosporidia.— Sporulation  ends  the  life  of  the  individual. 

Genus.  Species. 

Coccidium.  C.  cuniculi. 

Plasmodium.  P.  vivax. 

P.  malariae. 

P.  falciparum. 
Babesia.  B.  bigeminum. 

SuBPHYLUM  IV^     INFUSORIA.— Locomotion  by  means  of  cflia. 
Class  Ciliata. — Cilia  present  throughout  life. 

Getius.  Species. 

Balantidium.  B.  coli. 


SUBPHYLUM    SARCODINA 
Class  Rhizopoda 

These  are  protozoa  the  body  substance  of  which 
forms  changeable  protoplasmic  processes,  or  pseudo- 
podia,  for  the  taking  in  of  food  and  for  locomotion. 
They  possess  one  or  several  nuclei. 

I.  Genus  Entamoeba. — (i)  Entamoeba  Histolytica. — 
This  organism  is  found,  often  in  large  numbers,  in  the 
stools  of  tropical  dysentery  and  in  the  pus  and  walls 
of  hepatic  abscesses  associated  with  dysentery,  and 
is  generally  regarded  as  the  cause  of  the  disease.  It 
is  a  colorless,  granular  cell,  20  to  40  ^i  in  diameter 
(Fig.  no).  It  contains  one  or  more  distinct  vacuoles, 
a  round  nucleus,  which  ordinarily  is  obscured  by  the 
granules,  and  frequently  red  blood-corpuscles  and  bac- 
teria. When  at  rest  its  shape  is  spheric,  but  upon  a 
warm  slide  it  exhibits  the  characteristic  ameboid  motion, 
constantly  changing  its  shape  or  moving  slowly  about. 
This  motion  is  its  most  distinctive  feature.  If  neutral 
red  in  0.5  per  cent,  solution  be  run  under  the  cover-glass, 


PHYLUM  PROTOZOA 


329 


it  will  be  taken  up  by  the  amebae  and  other  protozoa 
and  render  them  conspicuous  without  killing  them 
("vital  staining"). 

When  the  presence  of  amebae  is  suspected,  the  stool 
should  be  passed  into  a  warm  vessel  and  kept  warm 
until  and  during  the  examination.  A  warm  stage  can 
be  improvised  from  a  plate  of  copper  with  a  hole  cut  in 
the  center.  This  is  placed  upon  the  stage  of  the  mi- 
croscope, and  one  of  the  projecting  ends  is  heated  with 


i 


Fig.  no. — Amoeba  coli  in  intestinal  mucus,  with  blood-corpuscles  and  bacteria  (Losch). 


a  small  flame.  Amebae  are  most  likely  to  be  found  in 
grayish  or  blood-streaked  particles  of  mucus.  Favor- 
able material  for  examination  can  be  obtained  at  one's 
convenience  by  inserting  into  the  rectum  a  large  catheter 
with  roughly  cut  lateral  openings.  A  sufficient  amount 
of  mucus  or  fecal  matter  will  usually  be  brought  away 
by  it. 

(2)  Other  Entamebae. — Entamceba  coli,  a  similar  but 
somewhat  smaller  organism  (10  to  20  fi),  with  less  dis- 


33©  ANIM.'VL  PARASITES 

tinct  pseudopodia  and  more  distinct  nucleus,  has  fre- 
quently been  found  in  the  stools  of  healthy  persons. 
E.  tetragena  has  recently  been  described.  It  apparently 
produces  a  chronic  diarrhea  and  is  not  confined  to  the 
tropics.  Another,  E.  buccalis,  has  been  found  in  decay- 
ing teeth  A  number  of  similar  organisms  have  been 
described  as  occurring  in  pus  and  in  ascitic  and  other 
body  fluids,  but  it  is  probable  that  in  many  cases,  at 
least,  the  structures  seen  were  ameboid  body  cells. 

SUBPHYLUM    MASTIGOPHORA    (FLAGELLATA) 
Qass  Zodmastigophora 

The  protozoa  of  this  subphylum  are  provided  with  one 
or  several  whip-like  appendages  with  lashing  motion, 
termed  flagella,  which  serve  for  locomotion  and,  in 
some  cases,  for  feeding.  They  generally  arise  from  the 
anterior  part  of  the  organism.  Some  members  of  the 
group  also  possess  an  undulating  membrane — a  delicate 
membranous  fold  which  extends  the  length  of  the  body, 
and  somewhat  suggests  a  fin.  When  in  active  motion 
this  gives  the  impression  of  a  row  of  cilia.  The  flagellata 
do  not  exhibit  ameboid  motion,  and,  in  general,  maintain 
an  unchanging  oval  or  spindle  shape,  and  contain  a  single 
nucleus.  The  cytoplasm  contains  numerous  granules 
and  usually  several  vacuoles,  one  or  more  of  which  may 
be  contractile.  Encystment  as  a  means  of  resisting 
unfavorable  conditions  is  common. 

I.  Genus  Spirochaeta. — The  spirochaetae  appear  to 
occupy  a  position  midway  between  the  bacteria  and 
protozoa,  but  are  more  frequently  described  with  the 
latter. 

(i)    Spirochaeta  Recurrentis. — This    spirochaite  was 


PHYLUM  PROTOZOA  33 1 

described  by  Obermeier  as  the  cause  of  relapsing  fever. 
It  appears  in  the  circulating  blood  during  the  febrile 
attack,  and,  unlike  the  malarial  parasite,  lives  in  the 
plasma  without  attacking  the  red  corpuscles.  The 
organism  is  an  actively  motile  spiral,  16  to  40  ;m  long, 
with  three  to  twelve  wide,  fairly  regular  turns.  It  can 
be  seen  in  fresh  unstained  blood  with  a  high  dry  lens, 
being  located  by  the  commotion  which  it  creates  among 


Fig.  III. — Spirochaete  of  relapsing  fever  (  X  looo)  (Karg  and  Schmorl). 

the  red  cells.  For  diagnosis,  thin  films,  stained  with 
Wright's  or  some  similar  blood-stain,  are  used  (Fig.  iii). 

Besides  Spirochceta  recurrentis,  a  number  of  distinct 
strains  have  been  described  in  connection  with  different 
types  of  relapsing  fever:  Sp.  novyi  (Plate  VII),  Sp.  kochi, 
Sp.  duUoni,  and  Sp.  carteri. 

(2)  Spirochaeta  Vincenti. — In  stained  smears  from  the 
ulcers  of  Vincent's  angina  (p.  380)  are  found  what 
appear  to  be  two  organisms.     One,  the  "  fusiform  bacil- 


332  ANIMAL  PARASITES 

lus/'  is  a  slender  rod,  6  to  12  /t/  long,  pointed  at  both 
ends  and  sometimes  curved.  The  other  is  a  slender 
spiral  organism,  30  to  40  fi  long,  with  three  to  eleven 
comparatively  shallow  turns  (Fig.  153).  These  were 
formerly  thought  to  be  bacteria,  a  spirillum  and  bacillus 
living  in  symbiosis.  The  present  tendency  is  to  regard 
them  as  stages  or  forms  of  the  same  organism,  and  to 
class  them  among  the  spirochaeta^.  The  same  organisms 
are  quite  constantly  present  in  large  numbers  in  ulcera- 
tive stomatitis  and  in  noma  They  are  not  infrequently 
found  in  small  numbers  in  normal  mouths. 


O 


Fig.  112. — Spiral   organisms:   A,   Treponema  pallidum;   B,  Spirochaeta  refringens;   C, 
Spirochaeta  dentium.     Two  red  corpuscles  are  also  shown  (  X  1200). 

(3)  Other  Spirochaetae. — A  number  of  harmless  forms 
are  of  interest  because  of  the  possibility  of  confusing 
them  with  the  more  important  pathogenic  varieties. 
Of  these,  Sp.  buccalis  and  Sp.  dentium  are  inhabitants 
of  the  normal  mouth.  The  former  is  similar  in  morphol- 
ogy to  Sp.  vincenti.  Sp.  dentium  (Fig.  112)  is  smaller, 
more  delicate,  has  deep  curves,  and  may  be  easily  mis- 
taken for  Treponema  pallidum.  It,  also,  stains  reddish 
with  Giemsa's  stain.  In  suspected  syphilitic  sores  of 
the  mouth  it  is,  therefore,  important   to  make  smears 


PHYLUM  PROTOZOA  333 

from  the  tissue  juices  rather  than  from  the  surface 
(see  p.  389).  Sp.  refringens  is  frequently  present  upon 
the  surface  of  ulcers,  especially  about  the  genitals,  and 
has  doubtless  many  times  been  mistaken  for  Treponema 
pallidum.  It  can  be  avoided  by  properly  securing  the 
material  for  examination;  but  its  morphology  should 
be  sufficient  to  prevent  confusion.  It  is  thicker  than 
the  organism  of  syphilis,  stains  more  deeply,  and  has 
fewer  and  shallower  curves  (Fig.  112).  Giemsa's  stain 
gives  it  a  bluish  color. 

2.  Genus  Treponema.— (i)  Treponema  Pallidum. — 
This  is  the  organism  of  syphilis.  Its  description  and 
methods  of  diagnosis  will  be  found  on  p.  388. 

(2)  Treponema  pertenue,  morphologically  very  similar 
to  Treponema  pallidum,  was  found  by  Castellani  in 
yaws,  a  skin  disease  of  the  tropics. 

3.  Genus  Trypanosoma. — Trypanosomes  have  been 
found  in  the  blood-plasma  of  a  great  variety  of  verte- 
brates. Many  of  them  appear  to  produce  no  symptoms, 
but  a  few  are  of  great  pathologic  importance.  As  seen 
in  the  blood,  they  are  elongated,  spindle-shaped  bodies, 
the  average  length  of  different  species  varying  from  10 
to  70  f£.  Along  one  side  there  runs  a  delicate  undulating 
membrane,  the  free  edge  of  which  appears  to  be  somewhat 
longer  than  the  attached  edge,  thus  throwing  it  into 
folds.  Somewhere  in  the  body,  usually  near  the  middle, 
is  a  comparatively  pale-staining  nucleus;  and  near  the 
posterior  end  is  a  smaller,  more  deeply  staining  chromatin 
mass,  the  micronucleus  or  blepharoplast.  A  number  of 
coarse,  deeply  staining  granules,  chromatophores,  may 
be  scattered  through  the  cytoplasm.  A  flagellum  arises 
in  the  blepharoplast,  passes  along  the  free  edge  of  the 


334  ANIMAL  PARASITES 

undulating  membrane,  and  is  continued  anteriorly  as  a 
free  flagellum.  These  details  of  structure  are  well 
shown  in  Plate  VII. 

The  life  history  of  the  trypanosomes  is  not  well  known. 
In  most  cases  there  is  an  alternation  of  hosts,  various 
insects  playing  the  part  of  definitive  host. 

Trypanosomes  have  been  much  studied  of  late,  and 
many  species  have  been  described.     Of  these,  only  a  few 


>  t 


/ 


Fig.  113. — Trypanosoma  lewisi  in  blood  of  rat.  The  red  corpuscles  were  decolorized 
with  acetic  acid  (X  1000)  (photograph  by  the  author  from  a  slide  presented  by  Prof. 

Novy). 

have  medical  interest.  At  least  two  have  been  found 
in  man. 

Trypanosoma  gamhiense  is  the  parasite  of  African 
"  sleeping  sickness."  Its  detection  in  the  blood  is 
described  on  p.  247. 

Trypanosoma  cruzi  is  a  small  form  which  has  been 
found  in  the  blood  of  man  in  Brazil. 

Trypanosoma  lewisi,  a  very  common  and  apparently 
harmless  parasite  of  gray  rats,  especially  sewer  rats,  is 


PHYLUM  PROTOZOA  335 

interesting  because  it  closely  resembles  the  pathogenic 
forms,  and  is  easily  obtained  for  study.  Its  posterior 
end  is  more  pointed  than  that  of  T.  gambiense. 

Trypanosoma  evansi,  T.  brucei,  and  T.  equiperdum 
produce  respectively  surra,  nagana,  and  dourine,  which 
are  common  and  important  diseases  of  horses,  mules, 
and  cattle  in  the  Philippines,  East  India,  and  Africa. 

4.  Genus  Leishmania. — The  several  species  which 
compose  this  genus  are  apparently  closely  related  to  the 
trypanosomes,  but  their  exact  classification  is  undeter- 
mined. They  have  been  grown  outside  the  body  and 
their  transformation  into  flagellated  trypanosome-like 
structures  has  been  demonstrated.  Calkins  places  them 
in  the  genus  Herpetomonas. 

(i)  Leishmania  donovani  is  the  cause  of  kala-azar,  an 
important  and  common  disease  of  India.  The  "  Leish- 
man-Donovan  bodies "  are  round  or  oval  structures, 
2  to  3  w  in  diameter,  with  two  distinct  chromatin  masses, 
one  large  and  pale,  the  other  small  and  deeply  staining. 
The  parasites  are  especially  abundant  in  the  spleen, 
splenic  puncture  being  resorted  to  for  diagnosis.  They 
are  readily  found  in  smears  stained  by  any  of  the  Roman- 
owsky  methods.  They  lie  chiefly  within  endothelial 
cells  and  leukocytes  They  are  also  present  within 
leukocytes  in  the  peripheral  blood,  but  are  difficult  to 
find  in  blood-smears. 

(2)  Leishmania  tropica  resembles  the  preceding.  It 
is  found,  lying  intracellularly,  in  the  granulation  tissue 
of  Delhi  boil  or  Oriental  sore. 

(3)  Leishmania  infantum  has  been  found  in  an  obscure 
form  of  infantile  splenomegaly  in  Algiers. 

5.  Genus  Cercomonas. — (i)  Cerccmonas  hominis  has 


33^ 


ANIMAL  PARASITES 


been  found  in  the  feces  in  a  variety  of  diarrheal  condi- 
tions, and  in  from  lo  to  25  per  cent,  of  healthy  persons  in 
tropical  regions.  It  is  probably  harmless.  The  body  is 
10  to  12  ^  long,  is  pointed  posteriorly,  and  has  a  flagel- 
lum  at  the  anterior  end  (Fig.  114).  The  nucleus  is 
difficult  to  make  out. 


"^^^g^Vr^ 


Fig.  114. — Cercoraonas  hominis  (about  X  500):  A,  Larger  variety;  B,  smaller  variety  • 

(Davaine). 

6.  Genus  Bodo. — (i)  Bodo  urinarius  is  sometimes  seen 
in  the  urine,  darting  about  in  various  directions.  It  is 
probably  an  accidental  contamination,  or  at  most  a 
harmless  invader.  It  has  a  lancet-shaped  body,  about 
10  u  long,  and  4s  somewhat  twisted  upon  itself,  with 
two  flagella  at  the  end. 


Fig.  115. — Trichomonas  vaginalis  (about  X  looo)  (after  Kolliker  and  Scanzoni). 

7.  Genus  Trichomonas.— (i)  Trichomonas  Vaginalis. 

— The  acid  discharge  of  catarrhal  vaginitis  sometimes 
contains  this  parasite  in  abundance.  It  is  oval  or  pear- 
shaped,  one  to  three  times  the  diameter  of  a  red  blood- 
corpuscle  in  length,  and  has  a  cluster  of  flagella  at  one 
end  (Fig.  115).     As  seen  in  fresh  material  it  is  not  unlike 


PHYLUM  PROTOZOA  337 

a  pus-corpuscle  in  size  and  general  appearance,  but  is 
actively  motile.  When  in  motion  the  flagella  are  not 
easily  seen.  No  pathogenic  significance  is  ascribed  to 
it  in  the  vagina,  but  a  few  cases  have  been  reported  in 
which  it  was  apparently  the  cause  of  a  urethritis  in  the 
male.     This  and  similar  organisms,  such  as  cercomonas 


Fig.  ii6. — Lamblia  intestinalis  from  the  intestines  of  a  mouse  (about  X  2000)  (Grass! 
and  Schweiakofi). 

and  bodo,  might  be  mistaken  for  spermatozoa  by  the 
totally  inexperienced  worker. 

(2)  Other  Trichomonads. — Various  forms  have  been 
described,  regarded  by  some  as  identical  with  T.  vagi- 
nalis, by  others  as  distinct  species.  Among  these  are 
T.  intestinalis,  sometimes  found  in  the  feces  in  diarrheal 
conditions,  and  T.  pulmonalis,  which  has  been  encoun- 
tered in  the  sputum  of  persons  suffering  from  pulmonary 
gangrene  and  putrid  bronchitis. 
22 


338  ANIMAL  PARASITES 

8.  Genus  Lamblia. — (i)  Lamblia  intestinalis  is  a  very 
common  parasite  in  the  tropics,  but  is  generally  consid- 
ered of  little  pathogenic  importance.  It  is  pear  shaped, 
measures  about  10  to  15  fJ.,  and  has  a  depression  on  one 
side  of  the  blunt  end,  by  which  it  attaches  itself  to  the 
tops  of  the  epithelial  cells  of  the  intestinal  wall.  Three 
pairs  of  flagella  are  arranged  about  the  depression  and 
one  pair  at  the  pointed  end  (Fig.  116). 

SUBPHYLUM    SPOROZOA 
Qass  Telosporidia 

All  the  members  of  this  class  are  parasitic,  but  only 
a  few  have  been  observed  in  man,  and  only  one  genus, 
Plasmodium,  is  of  much  importance  in  human  pathology. 
Propagation  is  by  means  of  spores,  and  sporulation  ends 
the  life  of  the  individual.  In  some  species  there  is  an 
alternation  of  generations,  in  one  of  which  sexual  proc- 
esses appear.  In  such  cases  the  male  individual  may 
be  provided  with  flagella.  Otherwise,  there  are  no 
special  organs  of  locomotion. 

I.  Genus  Coccidium. — (i)  Coccidium  cuniculi. — This 
is  a  very  common  parasite  of  the  rabbit  and  has  been 
much  studied;  but  extremely  few  authentic  cases  of 
infection  in  man  have  been  reported.  The  parasite, 
which  when  fully  developed  is  ovoid  in  shape  and 
measures  about  30  to  50  u  in  length  and  has  a  shell- 
like integument,  develops  within  the  epithelial  cells  of 
the  bile-passages.  Upon  reaching  adult  size  it  divides 
into  a  number  of  spores  or  merozoites  which  enter  other 
epithelial  cells  and  repeat  the  cycle.  A  sexual  cycle 
outside  the  body,  which  suggests  that  of  the  malarial 
parasite,  but  does  not  require  an  insect  host,  also  occurs. 


PHYLUM  PROTOZOA  339 

Infection  takes  place  from  ingestion  of  the   resulting 
sporozoites. 

2.  Genus  Plasmodium. — This  genus  includes  the  ma- 
larial parasites  which  have  already  been  described  (p.  248). 

3.  Genus  Babesia. — The  proper  position  of  this  genus 
is  uncertain.  It  is  placed  among  the  flagellates  by  some. 
The  chief  member  is  Babesia  bigeminum,  the  cause  of 
Texas  fever  in  cattle.  It  is  a  minute,  pear-shaped  or- 
ganism, lying  in  pairs  within  the  red  blood-corpuscles. 
An  organism,  B.  (or  Piroplasma)  hominis,  described  as 
occurring  in  the  red  cells  in  "  tick-fever  "  of  Montana,  is 
also  placed  in  this  genus,  but  its  pathogenicity  and  even 
its  existence  are  questionable. 

SUBPHYLUM   INFUSORU 
Qass  Qliata 

The  conspicuous  feature  of  this  class  is  the  presence 
of  cilia.  These  are  hair-like  appendages  which  have  a 
regular  to-and-fro  motion,  instead  of  the  irregular  lash- 
ing motion  of  flagella.  They  are  also  shorter  and  more 
numerous  than  flagella.  Most  infusoria  are  of  fixed 
shape  and  contain  two  nuclei.  Contractile  and  food- 
vacuoles  are  also  present.  Encystment  is  common. 
Only  one  species  is  of  medical  interest.  Certain  ciliated 
structures,  which  have  been  described  as  infusoria, 
notably  in  sputum  and  nasal  mucus,  were  probably 
ciliated  body  cells. 

1.  Genus  Balantidium. — (i)  Balantidium  Coli. — This 
parasite,  formerly  called  Paramcecium  coli,  is  an  occa- 
sional inhabitant  of  the  colon  of  man,  and  sometimes 
produces  diarrhea.  It  is  an  oval  organism,  about  o.i  mm. 
long,  is  covered  with  cilia,  and  contains  a  bean-shaped 


34°  ANIMAL  PAHASITES 

macronucleus,  a  globular  micronucleus,  two  contractile 
vacuoles,  and  variously  sized  granules  (Fig.  117). 


Fig.  117.— Balantidiumcoli  (about  X  300)  (after  Eichhorst). 

Its  ordinary  habitat  is  the  rectum  of  the  domestic 
pig,  where  it  apparently  causes  no  disturbance.  It 
probably  reaches  man  in  the  encysted  condition. 

PHYLUM  VERMIDEA 
Of  the  worms,  many  species  are  parasitic  in  man  and 
the  higher  animals.  In  some  cases  man  is  the  regular 
host;  in  others,  the  usual  habitat  is  some  one  of  the  ani- 
mals, and  the  occurrence  of  the  worm  in  man  is  more  or 
less  accidental.  Such  are  called  incidental  parasites. 
Only  those  worms  that  are  found  in  man  with  sufficient 
frequency  to  be  of  medical  interest  are  mentioned  here. 

PHAT.UM  \^RMIDEA 
SUBPHYLUM  I.     PLATYHELMINTHES.— Flat-worms. 
Class  Trematoda. — Flukes.     Unsegmented,  leaf  shaped. 
Genus.  Species. 

Fasciola.  F.  hepatica. 

Dicrocoelium.  D.  lanceatum. 

Opisthorchis.  Op.  felineus. 

Op.  sinensis. 
Paragonimus.  P.  westermani. 

Schistosomum.  S.  haematobium. 

S.  japonicum. 


PHYLUM  VERMIDEA 


Class  Cestoda. — Tapeworms.     Segmented,  ribbon  shaped. 


341 


Genus. 

Species. 

Taenia. 

T.  saginata. 

T.  solium. 

T.  echinococcus. 

Hymenolepis. 

H.  nana. 

Dipylidium. 

D.  caninum. 

Dibothriocephalus.    D.  latus. 

SuBPHYLUM  II.     NEMATHELMINTHES.— Round-worms. 
Class  Nematoda. — Unsegmented,  cylindric  or  fusiform. 


Genus. 

Species. 

Anguillula. 

A.  aceti. 

Ascaris. 

A.  lumbricoides. 

Oxyuris. 

0.  vermicularis. 

Filaria. 

F.  bancrofti. 

F.  philippinensis. 

F.  perstans. 

F.  diuma. 

F.  medinensis. 

Uncinaria. 

U.  duodenalis. 

Necator. 

N.  americanus. 

Strongyloides. 

S.  intestinalis. 

Trichinella. 

T.  spiralis. 

Trichocephalus. 

T.  trichiuris. 

SUBPHYLUM    PLATYHELMINTHES 
Class  Trematoda 

The  trematode  worms,  commonly  known  as  "  flukes," 
are  flat,  unsegmented,  generally  tongue-  or  leaf-shaped 
worms.  They  are  comparatively  small,  most  species 
averaging  between  5  and  15  mm.  in  length.  They  pos- 
sess an  incomplete  digestive  tract,  without  anus,  and  are 
provided  with  one  or  more  sucking  disks  by  means  of 
which  they  can  attach  themselves  to  the  host.  Some 
are  also  provided  with  booklets.  Nearly  all  species  are 
hermaphroditic,  and  the  eggs  of  nearly  all  are  operculated 
(provided  with  a  lid),   the  only  important  exception 


342  ANIMAL  PARASITES 

being  Schistosomum  hematobium,  the  egg  of  which  has  a 
characteristic  spine.  Development  takes  place  by  al- 
ternation of  generations,  the  intermediate  generation 
occurring  in  some  water  animal:  mollusks,  amphibians, 
fishes,  etc. 

1.  Genus  Fasciola. — (i)  Fasciola  Hepatica. — The 
"  liver  fluke  "  inhabits  the  bile-ducts  of  numerous  herbiv- 
orous animals,  especially  sheep,  where  it  is  an  important 
cause  of  disease.  It  brings  about  obstruction  of  the  bile- 
passages,  with  enlargement  and  degeneration  of  the  liver 


Fig.  ii8. — Fasciola  he[)atica,  about  two-thirds  natural  size  (Mosler  and  Peiper). 

— "  liver  rot."  A  species  of  snail  serves  as  intermediate 
host.  The  worm  is  leaf  shaped,  the  average  size  being 
about  2.8  by  1.2  cm.  The  anterior  end  projects  like  a 
beak  (head-cone  3  to  4  mm.  long)  (Fig.  118).  Ova  ap- 
pear in  the  feces.  They  are  yellowish  brown,  oval, 
operculated,  and  measure  about  0.13  by  0.07  mm. 

2.  Genus  Dicrocoelium. — (i)Dicrocoeliumlanceatum 
is  often  associated  with  the  liver  fluke  in  the  bile-passages 
of  animals,  but  is  neither  so  common  nor  so  widely 
distributed  geographically.  It  has  rarely  been  observed 
in  man.     It  is  smaller  (length  about  i  cm.)  and   more 


PHYLUM  VERMIDEA  343 

elongated.  The  long  diameter  of  the  eggs  is  about  0.04 
mm. 

3.  Genus  Opisthorchis. — (i)    Opisthorchis    felineus 

inhabits  the  gall-bladder  and  bile-ducts  of  the  domestic 
cat  and  a  few  other  animals.  Infection  in  man  has 
been  repeatedly  observed  in  Europe,  and  especially  in 
Siberia.  The  body  is  flat,  yellowish-red  in  color,  and 
almost  transparent.  It  measures  8  to  11  mm.  by  1.5  to 
2  mm.  The  eggs  are  oval,  with  a  well-defined  operculum 
at  the  narrower  end,  and  contain  a  ciliated  embryo  when 
deposited.     They  measure  about  30  by  11  fi. 

(2)  Opisthorchis  sinensis,  Uke  the  preceding  fluke, 
inhabits  the  gall-bladder  and  bile-ducts  of  domestic 
cats  and  dogs.  It  is,  however,  much  more  frequent  in 
man,  being  a  common  and  important  parasite  in  certain 
parts  of  Japan  and  China.  The  number  present  may 
be  very  great;  over  40CK)  were  counted  in  one  case. 
The  parasite  resembles  Op.  felineus  in  shape  and  color. 
It  is  10  to  14  mm.  long  and  2.5  to  4  mm.  broad.  The 
eggs  have  a  sharply  defined  lid  and  measure  27  to  30  by 
15  to  17  /[/.  When  they  appear  in  the  feces  they  contain 
a  ciliated  embryo.     The  intermediate  host  is  unknown. 

4.  Genus  Paragonimus. — (i)  Paragonimus  wester- 
mani,  called  the  "  lung  fluke,"  is  also  a  common  parasite 
of  man  in  Japan,  China,  and  Korea.  It  is  likewise  found 
in  dogs,  cats,  and  pigs  in  these  countries,  and,  according 
to  Ward  and  Stiles,  in  North  America  also.  It  inhabits 
the  lung,  causing  the  formation  of  small  cavities.  Mod- 
erate hemoptysis  is  the  principal  symptom.  Ova  are 
readily  found  in  the  sputum;  the  worms  themselves  are 
seldom  seen,  except  postmortem.  The  worms  are  faint 
reddish-brown  in  color,  egg  shaped  with  the  ventral 


344  ANIMAL  PARASITES 

surface  flattened,  and  measure  8  to  lo  mm.  by  4  to  6  mm. 
The  ova,  which  are  found  in  the  sputum,  are  thin  shelled, 
brownish  yellow,  and  average  about  0.093  by  0.057  n^™- 
Little  is  known  of  the  development  outside  the  body. 

5.  Genus  Schistosomum. — (i)  Schistosomum  Haema- 
tobium.— This  trematode,  frequently  called  Bilharzia 
hcematobia,  is  an  extremely  common  cause  of  disease 
(bilharziasis  or  Egyptian  hematuria)  in  northern  Africa, 
particularly  in  Egypt. 

Unlike  the  other  flukes,  the  sexes  are  separate.  The 
male  is  12  to  14  mm.  long  and  i  mm.  broad.  The  body 
is  flattened  and  the  lateral  edges  curl  ventrally,  forming  a 
longitudinal  groove,  in  which  the  female  lies  (Fig.  119). 


Fig.  iig. — Schistosomum  hccmatobium,  male  and  female  (about  X  4)  with  eggs  (about 
X  70)  (von  Jaksch). 

The  latter  is  cylindric  in  shape,  about  20  mm.  long  and 
0.25  mm.  in  diameter.  The  eggs  are  an  elongated  oval, 
about  0.15  mm.  long,  yellowish  in  color,  and  slightly 
transparent.  They  possess  no  lid,  such  as  characterize 
the  eggs  of  most  of  the  trematodes,  but  are  provided 
with  a  thorn-like  spine  which  is  placed  at  one  end  or 
laterally  near  the  end. 

In  man  the  worm  lives  in  the  veins,  particularly  the 
portal  vein  and  the  veins  of  the  bladder  and  rectum,  lead- 
ing to  obstruction  and  inflammation.  The  eggs  penetrate 
into  the  tissues  and  are  present  in  abundance  in  the 
mucosa  of  the  bladder  and  rectum.  They  also  appear  in 
the  urine  and  feces.     The  mode  of  infection  is  unknown. 


PHYLUM  VERMIDEA  345 

(2)  Schistosomum  japonicum  resembles  the  preceding 
morphologically,  but  both  the  male  and  female  are 
smaller.  The  ova  present  no  spines  and  somewhat  re- 
semble those  of  Uncinaria  duodenalis.  It  was  discov- 
ered in  Japan  in  1904  and  is  apparently  common  in  that 
country.     It  probably  inhabits  the  arteries. 

Qass  Cestoda 

The  cestodes,  or  tapeworms,  are  very  common  para- 
sites of  both  man  and  the  animals.  In  the  adult  stage 
they  consist  of  a  linear  series  of  flat,  rectangular  segments 
(proglottides),  at  one  end  of  which  is  a  smaller  segment, 
the  scolex  or  head,  especially  adapted  by  means  of  suck- 
ing discs  and  booklets  for  attachment  to  the  host. 
The  series  represents  a  colony,  of  which  the  scolex  is 
ancestor.  The  proglottides  are  sexually  complete  in- 
dividuals (in  most  cases  hermaphroditic),  which  are 
derived  from  the  scolex  by  budding.  With  the  excep- 
tion of  the  immature  segments  near  the  scolex,  each 
contains  a  uterus  filled  with  ova. 

The  large  tapeworms,  Tcenia  saginata,  T.  solium,  and 
Dihothriocephalus  latus,  are  distinguished  from  one  an- 
other mainly  by  the  structure  of  the  scolex  and  the 
uterus.  The  scolex  should  be  studied  with  a  low- 
power  objective  or  a  hand  lens.  The  uterus  is  best 
seen  by  pressing  the  segment  out  between  two  plates 
of  glass. 

All  the  tapeworms  pass  a  larval  stage  in  the  tissues  of 
an  intermediate  host,  which  is  rarely  of  the  same  species 
as  that  which  harbors  the  adult  worm.  From  the  ova 
which  have  developed  in  the  proglottides  of  the  adult 
worm,  and  which  pass  out  with  the  feces  of  the  host, 


346 


ANIMAL  PARASITES 


there  develop  embryos,  or  oncospheres,  each  provided 
with  three  pairs  of  horny  hooklets.  When  the  oncosphere 
is  taken  into  the  intestines  of  a  suitable  animal,  it  pene- 
trates to  the  muscles  or  viscera  and  there  forms  a  cyst 
in  which  develop  usually  one,  but  sometimes  many, 
scoHces,  which  are  identical  with  the  head  of  the  adult 
worm.  When  the  flesh  containing  this  cystic  stage  is 
eaten  without  sufficient  cooking  to  destroy  the  scolices, 
the  latter  attach  themselves  to  the  intestinal  wall  and 
produce  adult  tapeworms  by  budding. 


iglllllil"""^'  iii"-iTr||iiiiiimin 


Fig.  I20. — Taenia  saginata  (Eichhorst). 


Ordinarily,  only  the  adult  stage  occurs  in  man.  In  the 
case  of  TcEfiia  echinococcus  only  the  larval  stage  is  found. 
T.  saginata  and  T.  solium  may  infect  man  in  either  stage, 
although  the  cystic  stage  is  very  rare. 

Since  the  head,  or  scolex,  is  the  ancestor  from  which 
the  worm  is  formed  in  the  intestine,  it  is  important, 
after  giving  a  vermifuge,  to  make  certain  that  the  head 
has  been  passed  with  the  worm.  Should  it  remain,  a 
new  worm  will  develop. 

The  principal  tapeworms  found  in  man  belong  to 
the  genera  Taenia,  H>Tnenolepis,  and  Dibothriocephalus. 


PHYLUM  VERMIDEA  347 

1.  Genus  Taenia. — (i)  Taenia  Saginata  or  T.  Medio- 
canellata  (Fig.  120). — This,  the  beef  tapeworm,  is  the 
common  tapeworm  of  the  United  States.  Its  length 
sometimes  exceeds  twenty-five  feet.  The  middle  seg- 
ments measure  about  6  by  15  mm.  The  scolex  is 
about  the  size  of  a  pin-head,  and  is  surrounded  by  four 
sucking  discs,  but  has  no  booklets  (Fig.  122).  The  uterus 
extends  along  the  midline  of  the  segment  and  gives 
off  about  twenty  branches  upon  each  side  (Fig.  129). 


Fig.  121. — Eggs  of  Taenia  saginata,  magnifications  loo,  250,  and  500  diameters  (photo- 
graphs by  the  author). 

The  larval  stage  is  passed  in  the  muscles  of  various 
animals,  especially  cattle. 

The  scolex  is  ingested  with  the  meat,  its  capsule  is 
dissolved  by  the  digestive  juices,  and  it  attaches  itself  to 
the  intestinal  wall  by  means  of  its  suckers.  It  then 
develops  into  the  mature  worm. 

The  ova  are  present  in  the  stools  of  infected  persons, 
often  in  great  numbers  They  are  spheric  or  ovoid, 
yellow  in  color,  and  have  a  thick,  radially  striated  shell 
(Fig.  121).  Their  greatest  diameter  is  30  to  40  (J^  (about 
four  or  five  times  the  diameter  of  a  red  blood-corpuscle) . 


348  ANIMAL  PARASITES 

Vegetable  cells,  which  are  generally  present  in  the  feces, 
are  often  mistaken  for  them. 

(2)  Taenia  solium,  the  pork  tapeworm,  is  very  rare 
in  this  country.  It  is  usually  much  shorter  than  Tania 
saginata.  The  scolex  is  surrounded  by  four  sucking 
discs,  and  has  a  projection,  or  rostellum,  with  a  double 
row  of  horny  booklets  (Fig.  123).  The  uterus  has  only 
seven  to  ten  branches  (Fig.  129). 


Fig.  122. — Head  of  Taenia  saginata  (Mos-        Fig.  123. — Head  of  Tsenia  solium  (Mosler 
ler  and  Peiper).  and  Pe-per). 


The  cysticercus  stage  occurs  ordinarily  in  the  muscles 
of  the  pig,  but  is  occasionally  seen  in  man,  most  fre- 
quently affecting  the  brain  and  eye  {Cysticercus  celluloses). 

The  ova  closely  resemble  those  of  Tcenia  saginata,  but 
are  a  little  smaller  (Fig.  130). 

(3)  Taenia  Echinococcus. — The  mature  form  of  this 
tapeworm  inhabits  the  intestines  of  the  dog  and  wolf. 
The  larvae  develop  in  cattle  and  sheep  ordinarily,  but  are 
sometimes  found  in  man,  where  they  give  rise  to  echino- 
coccus or  "  hvdatid"  disease.    The  condition  is  unusual 


PHYLTJM  VEkMIDEA 


349 


in  America,  but  is  not  infrequent  in  Central  Europe  and 
is  common  in  Iceland  and  Australia. 

The  adult  parasite  is  2.5  to  5  mm.  long  and  consists 
of  only  four  segments  (Fig.  124).  It  contains  many  ova. 
When  the  ova  reach  the  digestive  tract  of  man  the  em- 
bryos are  set  free  and  find  their  way 
to  the  liver,  lung,  or  other  organ, 
where  they  develop  into  cysts,  thus 
losing  their  identity.  The  cysts  may 
attain  the  size  of  a  child's  head. 
Other  cysts,  called  "  daughter-cysts," 
are  formed  within  these.  The  cyst- 
wall  is  made  up  of  two  layers,  from 
the  inner  of  which  develop  larvae 
which  are  identical  with  the  head,  or 
scolex,  of  the  mature  parasite.  These 
are  ovoid  structures  0.2  to  0.3  mm. 
long.  Each  has  four  lateral  suckers 
and  a  rostellum  surmounted  by  a  double  circular  row 
of  horny  booklets.  The  rostellum  with  its  booklets  is 
frequently  invaginated  into  the  body. 

Diagnosis  of  echinococcus  disease  depends  upon  de- 
tection of  scolices,  free  booklets  which  have  fallen  off 
from  degenerated  scolices,  or  particles  of  cyst- wall,  which 
is  characteristically  laminated  and  usually  has  curled 
edges.  The  lamination  is  best  seen  at  the  torn  edge  of 
the  membrane.  These  can  be  found  in  fluid  withdrawn 
from  the  cysts  or,  less  frequently,  in  the  sputum  or  the 
urine,  when  the  disease  involves  the  lung  or  kidney  (Figs. 
•59 and  125).  The  cysts  are  sometimes  "barren,"  grow- 
ing to  a  considerable  size  without  producing  scolices. 

The  cyst  fluid  is  clear,  between  1.009  ^^^  i-oi5  in 


Fig.  124. — Taenia  echi- 
nococcus; enlarged  (Mos- 
ler  and  Peiper). 


350  ANIMAL  PARASITES 

specific  gravity,  and  contains  a  notable  amount  of  sodium 
chlorid,  but  no  albumin. 
2.  Genus  Hymenolepis.— (i)  Hymenolepis  nana,  the 

dwarf  tapeworm  (Fig.  126),  is  i  to  1.5  cm.  in  length 
and  0.5  to  0.7  mm.  in  breadth  at  the  widest  part.  The 
head  is  globular  and  has  a  rostellum  with  a  crown  of  24 


Fig.  125. — Scoles  and  booklets  of  Taenia  echinococcus  in  fluid  from  hepatic  cyst  (X300) 
(photographs  by  the  author). 

to  30  hooklets.  There  are  about  150  segments  The  eggs 
are  round  or  oval,  30  to  40  u  in  diameter,  and  resemble 
those  of  TcEuia  saginata.  The  worm  is  common  in  Europe 
and  America.  It  is  most  frequent  in  children  and  is  gen- 
erally present  in  large  numbers,  producing  considerable 
digestive  and  ner\-ous  disturbances.  The  mode  of  in- 
fection is  unknown. 


PHYLUM  VERMIDEA  r    ,  r- r-  ^"^I 

3.  Genus    Dipylidium.— t^y    Dipylidrum  r^cT^ipfn^p^,/; 
sometimes  called  Tania  g//i^i?Jci,ill$,fVp;:3f  cpiiqan(ion,l^ap^-v- 
worm  of  dogs  and  cats.     It  is  about  20  cm.  long  and  2  to 
3  mm.  broad.     The  intermediate  host  is  the  flea  or 
louse.     Infection  of  human  beings  is  not  common,  and 
is  mostly  confined  to  children,  who  are 
probably  infected  from  the  dog  licking 
their  mouths  or  from  getting  lice  or  fleas 
into  their  mouths. 

4.  Genus  Dibothriocephalus. — (i) 
Dibothriocephalus  latus,  the  fish  tape- 
worm, sometimes  reaches  fifty  feet  in 
length,  although  it  is  generally  not  more 
than  half  so  long.  When  several  worms 
are  present,  they  are  much  smaller.  It 
is  common  in  some  countries  of  Europe,  especially  Ire- 
land, and  in  Japan,  but  is  very  rare  in  this  country, 


nu 


Fig.  126. — Hymen- 
olepis  nana,  about 
natural  size  (Mosler 
and  Peiper). 


Fig.  127. — Head  of  Dibothriocephalus  latus  (about  Xg):  a,  a,  Head  grooves;  6,  neck 

(Blanchard). 

The  head  is  about  i  mm.  broad  and  is  not  unlike  the 
bowl  of  a  spoon  in  shape.  It  is  unprovided  with  either 
suckers  or  booklets,  but  has  two  longitudinal  grooves 
which  serve  the  same  purpose  (Fig.  127)  The  length  of 
the  segments  is  generally  less  than  their  breadth,  mature 
segments  measuring  about  3  by  10  or  12  mm.  The 
uterus,  which  is  situated  in  the  center  of  the  segment, 
is  roset  shaped  (Fig.  129)  and  brown  or  black  in  color. 
The  larval  stage  is  found  in  fish,  especially  the  pike. 


352 


ANIMAL  PARASITES 


• 


Fig.  128. — Ova  of  Dibothriocephalus  latus  (X  250  and  scx>).    The  lids  were  forced  open 
by  pressure  upon  the  cover-glass  (photographs  by  the  author). 


Fig.   129. — Segments  of — ft)  Tsnia  saginata;  (2)  Dibothriocephalus  latus;  (3)  Taenia 
solium,  showing  arrangement  of  uterus. 


The  ova  are  characteristic.     They  measure  about  45 
by  70  ^,  are  brown  in  color,  and  are  filled  with  small 


PHYLUM  VERMIDEA 


353 


spherules.  The  shell  is  thin  and  has  a  small  hinged  lid 
at  one  end.  As  the  eggs  appear  in  the  feces  the  Ud  is 
not  easily  seen,  but  it  may  be  demonstrated  by  sufl&cient 
pressure  upon  the  cover-glass  to  force  it  open  (Fig.  1 28) . 
The  only  other  operculated  eggs  met  with  in  man  are 
those  of  the  fluke- worms. 


a  b  c  d  e 

Fig.  130. — Comparative  size  of  eggs  of  intestinal  parasites  (about  X400):  a.  Taenia 
solium;  b.  Taenia  saginata;  c,  Ascaris  lumbricoides;  d,  Trichocephalus  trichiurus;  e, 
Oxyuris  vermicularis  (after  Striimpell). 

Dihothriocephalus  latus  is  interesting  clinically  because 
it  often  causes  a  very  severe  grade  of  anemia,  which  may 
be  indistinguishable  from  pernicious  anemia. 


SUBPHYLUM  NEMATHELMINTHES 
Qass  Nematoda 

The  nematodes,  or  round- worms,  are  cyhndric  or  fusi- 
form worms,  varying  in  length,  according  to  species, 
from  I  mm,'  to  40  or  80  cm.  As  a  rule,  the  sexes  are 
separate.  The  male  is  smaller  and  more  slender  than 
the  female.  In  a  few  cases  the  female  is  viviparous;  in 
most  cases  she  deposits  ova  which  are  characteristic, 
'so  that  the  finding  of  a  single  egg  may  establish  the 
diagnosis.  Except  in  a  few  instances  the  young  are 
different  from  the  adult,  and  must  pass  a  certain  larval 
stage   of  development  before   again   reaching  a   host. 

23 


354 


ANIMAL  PARASITES 


An  intermediate  host  is,  however,  necessary  with  only 
a  few  species. 

1.  Genus  Anguillula. — (i)    Anguillula  Aceti. — This 

worm,  commonly  called  the  "vinegar  eel,"  is  usually 
present  in  vinegar.  A  drop  of  the  vinegar,  particularly 
of  the  sediment,  will  frequently  show  great  numbers,  all 
in  active  motion:  males,  about  i  or  1.5  mm.  long;  females, 

somewhat  larger  and  fre- 
quently containing  several 
coiled  embryos;  and  young, 
of  all  sizes  up  to  the  adult 
(Fig.  60). 

The  vinegar  eel  is  never 
parasitic,  but  is  occasion- 
ally met  with  as  a  contami- 
nation in  the  urine  (see  p. 
171),  and  has  there  been 
mistaken  for  the  larva  of 
filaria  or  strongyloides. 

2.  Genus   Ascaris. — (i) 
Ascaris      Lumbricoides. — 
The  female  is  20  to  40  cm. 
long  and  about  6  mm.  thick 
(Fig.  131);  the  male,  a  little 
more    than   half   as  large 
Their  color  is   reddish   or 
brown.    They  are  the  com- 
mon   "  round- worms  "    so 
frequently    found    in   children.      Their   habitat  is   the 
small  intestine.     Large  numbers  are  sometimes  present. 
The  diagnosis  is  made  by  detection  of  the  worms  or  ova 
in  the  feces.     The  latter  are  generally  numerous.    They 


Fig.  131. 


-Ascaris   lumbricoides   (female) 
(Mosler  and  Peiper). 


PHYLUM  VERMIDEA 


355 


are  elliptic,  measuring  about  50  by  70  |U,  and  have  an  un- 
segmented  protoplasm  (Fig.  132).  The  shell  is  thick 
and  is  surrounded  by  an  uneven  gelatinous  envelop  which 
is  often  stained  with  bile. 

The  eggs  do  not  hatch  in  the  intestine  of  the  original 
host.  They  pass  out  in  the  feces  and,  after  a  variable 
period,  usually  about  five  weeks,  come  to  contain  an 
embryo  which  remains  within  the  shell  until  ingested 
by  a  new  host.     The  embryo  is  very  resistant  and  may 


Fig.  132. — Ova  of  Ascaris  lumbricoides  (X2S0  and  500)  (photographs  by  the  author). 


remain  alive  within  the  shell  for  years.  Upon  reaching 
the  intestine  of  the  new  host  it  hatches  out  and  develops 
into  the  adult  worm. 

3.  Genus  Oxyuris. — (i)  Oxyuris  Vermicularis. — This 
is  the  "thread- worm"  or  "pin-worm"  which  inhabits 
'the  colon  and  rectum,  especially  of  young  children.  Its 
presence  should  be  suspected  in  all  unexplained  cases  of 
pruritus  ani.  The  female  is  about  i  cm.  long;  the  male, 
about  0.6  cm.  (Fig.  133). 


356  ANIMAL  PARASITES 

The  worms  are  not  infrequently  found  in  the  feces;  the 
ova,  rarely.  The  latter  are  best  found  by  scraping  the 
skin  at  the  margin  of  the  anus,  where  they  are  deposited 
by  the  female,  who  wanders  out  from  the  rectum  for  this 
purpose,  this  producing  the  troublesome  itching.  They 
are  asymmetrically  oval  with  one  flattened  side,  are  about 
50  u  in  length,  and  often  contain  a  partially  developed 
embryo.  The  diagnosis  is  best  made  by  giving  a  pur- 
gative and  searching  the  stool  for  the  adult  worms. 

Infection  takes  place  through  swallowing  the  ova. 
Auto-infection  is  likely  to  occur  in  children;  the  ova 
cling  to  the  fingers  after  scratching  and  are  thus  carried 
to  the  mouth. 


Fig.  133. — Oxyuris  vermicularis  and  egg:  a.  Male  and  female,  natural  size;  b,  egg  (about 
X  250)  (after  Heller). 


4.  Genus  Filaria.— (i)  Filaria  Bancrofti.— The  adults 
are  thread-like  worms,  the  male  about  4  cm.,  the  female 
about  8  cm.,  long.  They  live  in  pairs  in  the  l>Tnphatic 
channels  and  glands,  especially  those  of  the  pelvis  and 
groin,  and  often  occur  in  such  numbers  as  to  obstruct 
the  flow  of  lymph.  This  is  the  most  common  cause  of 
elephantiasis.  Infection  is  very  common  in  tropical 
countries,  especially  in  Samoa,  the  West  Indies,  Central 
America,  and  the  Isthmus  of  Panama.  It  is  said  that  in 
Samoa  50  per  cent,  of  the  natives  are  infected. 

The  female  is  viviparous,  and  produces  vast  numbers 


PHYLUM  VERMIDEA  357 

of  embryos,  which  appear  in  the  circulating  blood.  The 
name  Filaria  sanguinis  hominis,  which  is  commonly 
applied  to  them,  is  incorrect,  since  they  do  not  consti- 
tute a  species.  These  embryos  are  about  as  wide 
as  a  red  corpuscle  and  0.2  to  0.4  mm.  long  (Fig.  99), 
and  are  very  actively  motile.  They  are  found  in 
the  peripheral  blood  only  at  night,  appearing  about  8 
P.  M.,  and  reaching  their  maximum  number — which  is 


Fig.  134. — Embryo  of  Filaria  bancrofti  in  chylous  hydrocele  flmd;  length,  zoo  /n; 
width,  8  >i.  A  number  of  red  blood-corpuscles  also  appear  (studied  through  courtesy 
of  Dr.  S.  D.  Van  Meter). 


sometimes  enormous — about  midnight.  If  the  patient 
change  his  time  of  sleeping,  they  will  appear  during  the 
day.  Infection  is  carried  by  a  variety  of  mosquito, 
which  acts  as  intermediate  host.  Diagnosis  rests  upon 
detection  of  embryos  in  the  blood,  as  described  on  p.  256. 
The  embryos  are  sometimes  found  in  urine  and 
chylous  fluids  from  the  serous  cavities.  Their  motion  is 
then  usually  less  active  than  when  in  blood.  That  shown 
in  Fig.  134  was  ahve  sixty  hours  after  removal  of  the 


35^  ANIMAL  P.\RASITES 

fluid.  Embryos  were  present  in  the  blood  of  the  same 
patient. 

A  number  of  other  filariae  whose  larvse  appear  in  the 
blood  are  known,  some  of  them  only  in  the  larval  stage. 
Among  these  are  Filaria  philippinensis  and  F.  perstans, 
which  exhibit  no  periodicity,  and  F.  diiirna  and  F.  loa, 
whose  embryos  appear  in  the  blood  during  the  day. 
The  adult  of  the  last  named  is  especially  frequent  in  the 
orbit  and  beneath  the  conjunctiva. 

(2)  Filaria  medinensis,  the  "guinea-worm,"  is  a  very 
interesting  and  important  worm  of  Africa  and  southern 
Asia.  It  is  thought  to  be  the  "  fiery  serpent  "  which 
molested  the  Children  of  Israel  in  the  Wilderness. 

The  larva  probably  enters  the  body  through  the  skin 
or  gastro-intestinal  tract.  It  wanders  about  in  the  sub- 
cutaneous tissues  until  maturity,  producing  slight,  if  any, 
symptoms.  The  male  has  only  recently  been  discovered. 
It  is  only  4  cm.  long.  It  dies  soon  after  the  female  is 
impregnated.  The  adult  female  is  a  very  slender, 
yellowish  worm,  about  50  to  80  cm.  long,  its  appearance 
somewhat  suggesting  a  catgut  suture.  When  gestation 
is  complete  the  greater  part  of  the  female's  body  consists 
of  a  uterus  filled  with  embryos.  The  female  then  travels 
to  the  feet  or  ankles  of  the  host  and  there  produces  a  red 
nodule  and,  finally,  an  ulcer,  from  the  center  of  which 
her  head  protrudes.  Through  this  great  numbers  of 
embryos  are  discharged  whenever  it  comes  in  contact 
with  water.  Little  damage  is  done  unless  the  worm  is 
pulled  out,  when  the  embryos  are  set  free  in  the  tissues 
and  cause  serious  disturbances. 

WTien  discharged  the  embryos  seek  out  a  small  crus- 
tacean, Cyclops,  which  serves  as  intermediate  host. 


PHYLUM  VERMIDEA 


359 


5.  Uncinaria  Duodenalis  and  Necator  Americanus. 

— These,  the  Old  and  the  New  World  hook-worm  res- 
pectively, are  among  the  more  harmful  of  the  animal 
parasites.  They  inhabit  the  small  intestine,  often  in 
great  numbers,  and  commonly  produce  a  severe  and  often 
fatal  anemia.  The  presence  of  a  few,  however,  may 
cause  slight,  if  any,  disturbance. 


Fig.  I3S- — Uncinaria  duodenalis:  a,  Male  (natural  size);  b,  female  (natural  size);  c,  male 
(enlarged);  d,  female  (enlarged);  e,  head;  /,  /,  /,  eggs  (after  v.  Jaksch). 

Uncinaria  duodenalis  is  common  in  southern  Europe 
and  in  Egypt.  The  body  is  cylindric,  reddish  in  color, 
and  the  head  is  bent  sharply.  The  oral  cavity  has 
six  hook-like  teeth.  The  female  is  12  to  18  mm.  long 
and  the  tail  is  pointed.  The  male  is  8  to  10  mm.  long  and 
the  posterior  end  is  expanded  into  an  umbrella-like  pouch, 
the  caudal  bursa.  The  eggs  are  oval  and  have  a  thin, 
smooth,  transparent  shell.  As  they  appear  in  the  feces 
the  protoplasm  is  divided  into  2,  4,  8,  or  more  rounded 


360 


ANIMAL  PARASITES 


segments  (Fig.  135).     They  measure  32  to  40  |t^  by  55  to 

Necator  americanus  is  very  common  in  subtropical 
America,  including  the  southern  part  of  the  United 
States  and  the  West  Indies.  In  Porto  Rico  90  per  cent, 
of  the  rural  population  is  infected.  Isolated  cases,  prob- 
ably imported,  have  been  seen  in  most  of  the  Northern 


Fig.  136. — Four  eggs  of  the  New  World  hook-woriu  ^iSccator  americanus),  in  the 
one-,  two-,  and  four-cell  stages.  The  egg  showing  three  cells  is  a  lateral  view  of  a  four- 
cell  stage  (about  X3S0)  (after  Stiles). 

States.  The  American  hook-worm  is  smaller  than  the 
Old  World  variety,  the  male  being  7  to  9  mm.  long,  the 
female  9  to  1 1  mm.  The  four  ventral  hook-Hke  teeth  are 
replaced  by  chitinous  plates.  There  are  also  differences 
in  the  caudal  bursa  of  the  male,  and  in  the  situation  of 
the  vulva  in  the  female.  The  ova  (Fig.  136)  resemble 
those  of  Uficinaria  duodenalis,  but  are  larger,  36  to  4.0^ 
by  67  to  75  y.. 
The  life-history  of  the  two  worms  is  probably  the  same. 


PHYLUM  VERMIDEA  36 1 

The  ova  pass  out  with  the  feces,  and,  under  favorable  con- 
ditions of  warmth  and  moisture,  develop  an  embryo 
which  hatches  within  a  few  days.  The  resulting  larvae 
pass  through  a  stage  of  development  in  warm  moist 
earth,  growing  to  a  length  of  0.5  to  0.6  mm.,  and  moulting 
twice.  They  are  then  ready  to  infect  a  new  host.  In 
some  cases  they  probably  reach  the  host's  intestine  by 
way  of  the  mouth,  with  food  or  water;  but  the  usual 
route  is  probably  that  established  by  Loos.  When  moist 
earth  containing  the  larvae  comes  in  contact  with  the 
skin,  they  penetrate  into  the  subcutaneous  tissues. 
This  is  favored  by  retention  of  mud  between  the  toes  of 
those  who  go  barefooted.  When  the  larvae  are  abundant 
a  dermatitis  is  induced  {"  ground  itch").  From  the  sub- 
cutaneous tissue  they  pass  by  way  of  lymph-  and  blood- 
streams to  the  lungs.  Here  they  make  their  way  into 
the  smaller  bronchi,  are  carried  by  the  bronchial  mucus 
to  the  pharynx,  and  are  swallowed.  They  thus  ulti- 
mately reach  the  small  intestine,  where  they  develop 
into  mature  worms. 

The  diagnosis  of  hook-worm  infection,  which  is  assum- 
ing increasing  importance  in  this  country,  must  rest  upon 
detection  of  ova  in  the  ^eces.  The  worms  themselves 
seldom  appear  except  after  thymol  and  a  cathartic.  A 
small  portion  of  the  feces,  diluted  with  water  if  necessary, 
is  placed  upon  a  slide,  covered,  and  searched  with  a  16 
mm.  objective.  A  higher  power  may  rarely  be  neces- 
.sary  to  positively  identify  an  egg,  but  should  not  be 
used  as  a  finder.  The  eggs  are  nearly  always  typic, 
showing  a  thin  but  very  distinct  shell,  a  clear  zone,  and 
a  segmented  protoplasm,  and  after  having  once  been  seen 
are  not  easily  mistaken.      In  severe  infections  eggs  may 


362  ANIMAL  PARASITES 

be  found  in  every  microscopic  field;  in  most  cases,  even 
though  comparatively  mild,  they  can  be  found  on  the  first 
slide  examined.  It  is  seldom  necessary  to  search  more 
than  half  a  dozen  slides.  When  they  are  scarce,  some 
method  of  sedimenting  the  feces  may  be  tried,  but  this 
is  rarely  necessary. 

6.  Genus  Strongyloides.— (i)  Strongyloides  Intes- 
tinalis. — Infection  with  this  worm  is  by  no  means  so  rare 
in  this  country  as  the  few  clinical  reports  would  indicate. 
It  is  very  common  in  subtropical  countries,  notably  in 
Italy  and  in  southern  China.  It  seems  probable  that 
the  parasite  is  the  cause  of  "  Cochin  China  diarrhea," 
although  some  authorities  regard  it  as  harmless. 

The  adult  worm,  which  reproduces  by  parthenogenesis, 
is  about  2  mm.  long.  It  inhabits  the  upper  portion  of 
the  small  intestine,  but  neither  it  nor  the  ova  appear  in 
the  stool  unless  an  active  diarrhea  exists.  Ordinarily 
the  eggs  hatch  in  the  intestines,  and  when  infection  is 
severe  embryos  can  be  found  in  the  feces  in  large  num- 
bers. These  are  the  ''  rhabditiform  embryos,"  which 
measure  about  0.40  by  0.02  mm.  They  are  actively 
motile,  and  are  best  found  by  making  a  small  depression 
in  the  fecal  mass,  filling  it  with  water,  and  keeping  in  a 
warm  place  (preferably  an  incubator)  for  twelve  to 
twenty-four  hours.  The  embryos  will  collect  in  the 
water,  and  can  be  easily  found  by  transferring  a  drop 
to  a  slide  and  examining  with  a  16  mm.  objective.  The 
inexperienced  worker  should  make  sure  that  the  worms 
move,  or  he  may  be  misled  by  the  vegetable  spines 
which  are  generally  present  in  the  feces. '  Certain  of 
these  spines  (notably  those  from  the  skin  of  a  peach) 
closely  resemble  small  worms. 


PHYLUM   VERMIDEA  363 

Outside  the  body  the  rhabditiform  embryos  develop 
into  a  free-Hving,  sexually  differentiated  generation.  The 
young  of  this  generation  are  the  more  slender  "filari- 
form embryos"  (Fig.  137).  Infection  can  occur  either 
through  these  embryos  of  the  free-living  generation  or  by 
direct  transformation  of  rhabditiform  into  filariform  em- 
bryos, and  these  into  the  parthenogenic  parasitic  adult. 


Fig.  137. — Strongyloides  intestinalis:  A,  Mature  female;  B,  rhabditiform  larva;  C,  filari- 
form larva  (after  Braun). 

7.  Genus    Trichinella. — (i)    Trichinella    Spiralis. — 

This  is  a  very  small  worm,  not  exceeding  3  mm.  in 
length  when  fully  developed.  Infection  in  man  occurs 
from  ingestion  of  insufficiently  cooked  pork,  which 
contains  encysted  embryos.  Ordinary  "curing"  of 
pork  does  not  kill  them.  These  reach  maturity  in 
the  small  intestine.  Soon  after  copulation  the  males 
die,  and  the  females  penetrate  into  the  mucous  mem- 
brane. They  live  in  this  situation  about  six  weeks, 
giving  birth  to  great  numbers  of  young,  averaging  as 
high  as  1500  from  a  single  female.  The  larvae  migrate 
to  the  striated  muscles,  chiefly  near  the  tendinous  inser- 
tions, where  they  grow  to  a  length  of  about  0.8  mm.,  and 
finally  become  encysted.  In  this  condition  they  may 
remain  alive  and  capable  of  developing  for  as  long  as 
twenty-five  years. 


364  ANIMAL  PARASITES 

Trichiniasis  is  generally  accompanied  by  a  marked 
eosinophilia.  The  diagnosis  is  made  by  teasing  out  upon 
a  slide  a  bit  of  muscle,  obtained  preferably  from  the  outer 
head  of  the  gastrocnemius,  the  insertion  of  the  deltoid, 


Fit;.  138. — Trichinella  spiralis  (larvae)  from  head  of  right  gastrocnemius  muscle;  seventh 
week  of  disease  (two-thirds  objective;  eye-piece  4)  (Boston). 

or  the  lower  portion  of  the  biceps.  The  coiled  embryos 
can  easily  be  seen  with  a  i6  mm.  objective  (Fig.  138). 
The  embryos  can  be  found  in  the  blood  (p.  257)  before 
they  have  reached  their  final  resting-place  in  the  muscles. 


Fis.   13Q. — Trichocephalus  trichiurus:  a.  Female;  b,  male  (natural  size)   (Heller). 

8.  Genus  Trichocephalus.— (i)  Trichocephalus  Tri- 
chiurus.— This,  the  "whip-worm,"  is  4  or  5  cm.  long. 
Its  anterior  portion  is  slender  and  thread-like,  while 
the    posterior    portion    is    thicker    (Fig.    139).      It    is 


PHYLUM   VERMIDE  365 

widely  distributed  geographically,  and  is  one  of  the  most 
common  of  intestinal  parasites  in  this  country.  It  lives 
in  the  large  intestine,  especially  the  cecum,  with  its 
slender  extremity  embedded  in  the  mucous  membrane. 
Whip- worms  do  not,  as  a  rule,  produce  any  symptoms, 
although  gastro-intestinal  disturbances,  nervous  symp- 
toms, and  anemia  have  been  ascribed  to  them.  They, 
as  well  as  many  other  intestinal  parasites,  are  probably 


Fig.  140. — Ova  of  Trichocephalus  trichiurus  ( X  250  and  500)  (photographs  by  the  author). 

an  important  factor  in  the  etiology  of  appendicitis, 
typhoid  fever,  and  other  intestinal  infections.  The 
damage  which  they  do  to  the  mucous  membrane  favors 
bacterial  invasion. 

The  number  present  is  usually  small.  The  worms 
themselves  are  rarely  found  in  the  feces.  The  ova,  which 
are  not  often  abundant,  are  easily  recognized.  They 
are  brown,  ovoid  in  shape,  about  50  fi  long,  and  have  a 
button-like  projection  at  each  end  (Fig.  140)- 


366  ANIMAL  PARASITES 

PHYLUM  ARTHROPODA 

The  arthropoda  which  are  parasitic  to  man  belong  to 
the  classes  Arachnoidea  and  Insecta.  They  are  nearly 
all  external  parasites,  and  the  reader  is  referred  to  the 
standard  works  upon  diseases  of  the  skin  for  descriptions. 
The  several  species  of  the  louse  {Pediculus  capitis,  P. 
vcslimenti,  P.  pubis),  the  itch  mite  {Sarcoptes  scahiei),  and 
the  small  organism  {Demodex  folliculorum)  which  lives 
in  the  sebaceous  glands,  especially  about  the  face,  are 
the  most  common  members  of  this  group. 

A  number  of  flies  may  deposit  their  ova  in  wounds  or  in 
such  of  the  body  cavities  as  they  can  reach,  and  the  re- 
sulting maggots  may  cause  intense  irritation.  Ova  may 
be  swallowed  with  the  food  and  the  maggots  appear  in  the 
feces.  Probably  most  important  is  the  "screw  worm," 
the  larva  of  Chrysomyia  macellaria,  infection  with  which 
is  not  rare  in  some  parts  of  the  United  States.  The  ova 
are  most  commonly  deposited  in  the  nasal  passages,  and 
the  larvae,  which  may  be  present  in  great  numbers, 
burrow  through  the  soft  parts,  cartilage,  and  even  bone, 
always  with  serious  and  often  with  fatal  results. 


CHAPTER  VII 

MISCELLANEOUS  EXAMINATIONS 

PUS 

Pus  contains  much  granular  debris  and  numerous  more 
or  less  degenerated  cells,  the  great  majority  being  poly- 
morphonuclear leukocytes — so-called  "pus-corpuscles." 
EosinophiHc  leukocytes  are  common  in  gonorrheal  pus 
and  in  asthmatic  sputum.  Examination  of  pus  is  di- 
rected chiefly  to  detection  of  bacteria. 

When  very  few  bacteria  are  present,  culture  methods, 
which  are  outlined  in  Chapter  VIII,  must  be  resorted  to. 
When  considerable  numbers  are  present,  they  can  be 
detected  and  often  identified  in  cover-glass  smears. 
Several  smears  should  be  made,  dried,  and  fixed,  as 
described  on  p.  407. 

One  of  these  should  be  stained  with  a  bacterial  stain, 
Loffler's  methylene-blue  and  Pappenheim's  pyronin- 
methyl-green  are  especially  satisfactory  for  this  pur- 
pose. These  stains  are  applied  for  one-half  minute 
to  two  minutes  or  longer,  without  heating;  the  prep- 
aration is  rinsed  in  water,  dried,  mounted,  and  examined 
with  an  oil-immersion  lens.  Another  smear  should  be 
stained  by  Gram's  method.  These  will  give  information 
concerning  all  bacteria  which  may  be  present,  and  fre- 
quently no  other  procedure  will  be  necessary  for  their 
identification. 

367 


368  MISCELLANEOUS   EXAMINATIONS 

The  most  common  pus-producing  organisms  are 
staphylococci  and  streptococci.  They  are  both  cocci,  or 
spheres,  their  average  diameter  being  about  i  ^.  Staphy- 
lococci are  commonly  grouped  in  clusters,  often  compared 
to  bunches  of  grapes  (Fig.  141).  There  are  several 
varieties  which  can  be  distinguished  only  in  cultures. 
Streptococci  are  arranged  side  by  side,  forming  chains 
of  variable  length  (Fig.  142).  Sometimes  there  are  only 
three  or  four  individuals  in  a  chain;  sometimes  a  chain 


Fig.  141. — Staphylococcus  pyogenes  albus  from  an  abscess  of  the  parotid  gland  (Jakob). 

is  so  long  as  to  extend  across  several  microscopic  fields. 
Streptococci  are  more  virulent  than  staphylococci,  and 
are  less  commonly  met.  Both  are  Gram-positive. 
Their  cultural  characteristics  are  given  on  p.  415. 

Should  bacteria  resembling  pneumococci  be  found, 
Buerger's  or  Smith's  method  for  capsules  (p.  55)  should 
be  tried.  When  these  are  not  available,  capsules  can 
usually  be  shown  by  the  method  of  Hiss.  The  dried 
and  fixed  smear  is  covered  with  a  stain  composed  of  5  c.c. 
saturated  alcoholic  solution  gentian-violet  and  95  c.c. 


PUS  369 

distilled  water,  and  heated  until  steam  rises.  The  prep- 
aration is  then  washed  with  20  per  cent,  solution  of 
copper  sulphate,  dried,  and  mounted  in  Canada  balsam. 

Pneumococci  may  give  rise  to  inflammation  in  many 
locations  (see  p.  54).  When  they  form  short  chains, 
demonstration  of  the  capsule  is  necessary  to  distinguish 
them  from  streptococci. 

If  tuberculosis  be  suspected,  the  smears  should  be 
stained  by  one  of  the  methods  for  the  tubercle  bacillus 


Fig.  142. — Streptococcus  pyogenes  from  a  case  of  empyema  (Jakob). 

(pp.  49  and  51),  or  guinea-pigs  may  be  inoculated. 
The  bacilli  are  generally  diflicult  to  find  in  pus,  and 
bacteria-free  pus  would  suggest  tuberculosis. 

Gonococci,  when  typic,  can  usually  be  identified  with 
sufficient  certainty  for  clinical  purposes  in  the  smear 
stained  with  Loffler's  methylene-blue  or,  much  better, 
Pappenheim's  pyronin -methyl-green.  They  are  coffee- 
bean-shaped  cocci  which  lie  in  pairs  with  their  flat  sur- 
faces together  (Fig.  144).  They  lie  for  the  most  part 
within  pus-cells,  an  occasional  cell  being  filled  with  them, 

24 


370 


MISCELLANEOUS   EXAMINATIONS 


while  the  surrounding  cells  contain  few  or  none.  A  few 
are  found  outside  of  the  cells.  It  is  not  usual  to  find 
gonococci  when  many  other  bacteria  are  present,  even 


Fig.  143. — Diplococcus  pneumoniae  from  ulcer  of  cornea  (obj.  one-twelfth  oil  immersion) 
(study  throuKh  courtesy  of  Dr.  C.  A.  Oliver)  (Boston). 

though  the  pus  is  primarily  of  gonorrheal  origin.  When- 
ever the  identity  of  the  organism  is  at  all  questionable, 
Gram's  method  should  be  tried.     In  rare  instances  it 


Fig.  144. — Gonococci  in  urethral  pus  (McFarland). 

may  be  necessary  to  resort  to  cultures.  The  gonococcus 
is  distinguished  by  its  failure  to  grow  upon  ordinary 
media  (see  p.  416). 


PERITONEAL,  PLEURAL,  AND  PERICARDIAL  FLUIDS   37 1 

Gonococci  are  generally  easily  found  in  pus  from  un- 
treated acute  and  subacute  gonorrheal  inflammations — 
conjunctivitis,  urethritis,  etc. — but  are  found  with  diffi- 
culty in  pus  from  chronic  inflammations  and  abscesses, 
and  in  urinary  sediments. 

PERITONEAL,  PLEURAL,  AND  PERICARDLA.L  FLUIDS 

The  serous  cavities  contain  very  little  fluid  normally, 
but  considerable  quantities  are  frequently  present  as  a 
result  of  pathologic  conditions.  The  pathologic  fluids  are 
classed  as  transudates  and  exudates. 

Transudates  are  non-inflammatory  in  origin.  They 
contain  only  a  few  cells,  and  less  than  2.5  per  cent,  of 
albumin,  and  do  not  coagulate  spontaneously.  The 
specific  gravity  is  below  1.018.  Micro-organisms  are 
seldom  present. 

Exudates  are  of  inflammatory  origin.  They  are  richer 
in  cells  and  albumin,  and  tend  to  coagulate  upon  stand- 
ing. The  specific  gravity  is  above  1.018.  Bacteria  are 
generally  present,  and  often  numerous.  The  amount  of 
albumin  is  estimated  by  Esbach's  method,  after  diluting 
the  fluid.  Bacteria  are  recognized  by  cultures,  animal 
inoculation,  or  stained  smears. 

Exudates  are  usually  classed  as  serous,  serofibrinous, 
seropurulent,  purulent,  putrid,  and  hemorrhagic,  which 
terms  require  no  explanation.  In  addition,  chylous  and 
chyloid  exudates  are  occasionally  met,  particularly  in  the 
peritoneal  cavity.  In  the  chylous  form  the  milkiness  is 
due  mainly  to  the  presence  of  minute  fat-droplets,  and  is 
the  result  of  rupture  of  a  lymph-vessel  usually  from 
obstruction  of  the  thoracic  duct.  Chyloid  exudates 
are  milky  chiefly  from  proteins  in  suspension,  or  fine 


372  MISCELLANEOUS   EXAMINATIONS 

debris  from  broken-down  cells.  These  exudates  are  most 
frequently  seen  in  carcinoma  and  tuberculosis  of  the 
peritoneum. 

Cytodiagnosis. — This  is  diagnosis  from  a  differential 
count  of  the  cells  in  a  transudate  or  exudate,  particularly 
one  of  pleural  or  peritoneal  origin. 

The  fresh  fluid,  obtained  by  aspiration,  is  centrifugal- 
ized  for  at  least  five  minutes;  the  supernatant  liquid  is 


Fig.  145. — Cytodiagnosis.  Polymorphonuclear  leukocytes  and  swollen  endothelial 
cells  from  acute  infectious  non-tuberculous  pleuritis  (Percy  Musgrave;  photo  by  L.  S. 
Brown). 

poured  off;  and  cover-glass  smears  are  made  and  dried  in 
the  air.  The  smears  are  then  stained  with  Wright's 
blood-stain,  to  which  one-third  its  volume  of  pure  methyl- 
alcohol  has  been  added.  Cover  the  smear  with  this  fluid 
for  one-half  minute,  then  dilute  with  8  or  10  drops  of 
water,  and  let  stand  about  two  minutes.  Wash  gently 
in  water,  and  dry  by  holding  the  cover-glass  between  the 
fingers  over  a  flame.  Mount  in  balsam  and  examine 
with  an  oil-immersion  objective. 


PERITONEAL,   PLEURAL,   AND  PERICARDIAL  FLUIDS      373 


Fig.  146. — Cytodiagnosis.     Lymphoid  cells  from  pleural  fluid;  case  of  tuberculous  pleuritis 
(Percy  Musgrave;  photo  by  L.  S.  Brown). 


Fig.    147. — Cytodiagnosis.     Endothelial  cells   from   transudate  or  mechanical   effusion 
(Percy  Musgrave;  photo  by  L.  S.  Brown). 

Predominance  of  polymorphonuclear  leukocytes  (pus- 
corpuscles)  points  to  an  acute  infectious  process  (Fig. 

145)- 


374  MISCELLANEOUS   EXAMINATIONS 

Predominance  of  lymphocytes  (Fig.  146)  generally  sig- 
nifies tuberculosis.  Tuberculous  pleurisy  due  to  direct 
extension  from  the  lung  may  give  excess  of  polymorpho- 
nuclears owing  to  mixed  infection. 

Predominance  of  endothelial  cells,  few  cells  of  any  kind 
being  present,  indicates  a  transudate  (Fig.  147).  Endo- 
thelial cells  generally  predominate  in  carcinoma,  but  are 
accompanied  by  considerable  numbers  of  lymphocytes 
and  red  blood-corpuscles. 

CEREBROSPINAL  FLUID 

Examination  of  the  fluid  obtained  by  lumbar  puncture 
is  of  value  in  diagnosis  of  certain  forms  of  meningitis. 

Tubercle  bacilli  can  be  found  in  the  majority  of  cases 
of  tuberculous  meningitis.  The  sediment,  obtained  by 
thorough  centrifugahzation  or  by  coagulation  and  treat- 
ment with  antiformin,  is  spread  upon  slides  and  stained 
by  one  of  the  methods  already  given.  A  consider- 
able number  of  smears  should  be  examined.  In 
doubtful  cases  inoculation  of  guinea-pigs  must  be  re- 
sorted to. 

The  Diplococcus  intracellularis  meningitidis  is  recog- 
nized as  the  cause  of  epidemic  cerebrospinal  fever,  and 
can  be  detected  in  the  cerebrospinal  fluid  of  most  cases, 
especially  those  which  run  an  acute  course.  Cover-glass 
smears  from  the  sediment  should  be  stained  by  the 
method  for  the  gonococcus  (p.  369).  The  meningo- 
coccus is  an  intracellular  diplococcus  which  often  cannot 
be  distinguished  from  the  gonococcus  in  stained  smears 
(Fig.  148).  It,  also,  decolorizes  by  Gram's  method.  The 
presence  of  such  a  diplococcus  in  meningeal  exudates  is, 
however,  sufficient  for  its  identification. 


ANIMAL  INOCULATION  375 

Various  organisms  have  been  found  in  other  forms  of 
meningitis — the    pneumococcus    most    frequently.     In 


Fig.  148. — Diplococcus  intracellularis  meningitidis  in  leukocytes  (X2000)  (Wright  and 

Brown). 

some  cases  no  micro-organisms  can  be  detected  even  by 
culture  methods. 

ANIMAL  INOCULATION 

Inoculation  of  animals  is  one  of  the  most  reliable  means 
of  verifying  the  presence  of  certain  micro-organisms  in 
fluids  and  other  pathologic  material,  and  is  helpful  in 
determining  the  species  of  bacteria  which  have  been 
isolated  in  pure  culture. 

Clinically,  it  is  applied  almost  exclusively  to  demon- 
stration of  the  tubercle  bacillus  when  other  means  have 
failed  or  are  uncertain.     The  guinea-pig  is  the  most 


376 


MISCELLANEOUS   EXAMINATIONS 


suitable  animal  for  this  purpose.  When  the  suspected 
material  is  fluid  and  contains  pus,  it  should  be  well  cen- 
trifugalized,  and  one  or  two  cubic  centimeters  of  the 
sediment  injected  by  means  of  a  large  hypodermic  needle 
into  the  peritoneal  cavity  or  underneath  the  loose  skin  of 
the  groin.  Fluids  from  which  no  sediment  can  be  ob- 
tained must  be  injected  directly  into  the  peritoneal  cavity, 
since  at  least  lo  c.c.  are  required,  which  is  too  great  an 


Fig.  i4g. — Influenza  bacilli  in  spinal  fluid.     Case  of  meningitis  (X  looo)  (photograph  by 

the  author). 

amount  to  inject  hypodermically.  Sohd  material  should 
be  placed  in  a  pocket  made  by  snipping  the  skin  of  the 
groin  with  scissors,  and  freeing  it  from  the  underlying 
tissues  for  a  short  distance  around  the  opening.  When 
the  intraperitoneal  method  is  selected,  several  animals 
must  be  inoculated,  since  some  are  likely  to  die  from 
peritonitis  caused  by  other  organisms  before  the  tubercle 
bacillus  has  had  time  to  produce  its  characteristic  lesions. 
The  animals  should  be  killed-  at  the  end  of  six  or  eight 


THE   MOUTH  377 

weeks,  if  they  do  not  die  before  that  time,  and  a  careful 
postmortem  examination  should  be  made  for  the  char- 
acteristic pearl-gray  or  yellow  tubercles  scattered  over 
the  peritoneum  and  through  the  abdominal  organs,  par- 
ticularly the  spleen,  and  for  caseous  inguinal  and  retro- 
peritoneal lymph-glands.  The  tubercles  and  portions  of 
the  caseous  glands  should  be  crushed  between  two  slides, 
dried,  and  stained  for  tubercle  bacilli.  The  bacilli  are 
difficult  to  find  in  the  caseous  material. 

THE  MOUTH 
Micro-organisms  are  always  present  in  large  numbers. 
Among  these  is  Leptothrix  huccalis  (Fig.  150),  which  is 


Fig.  150. — Gingival  deposit  (iinstained):  a.  Squamous  epithelial  cells;  b,  leujiocs^tes;  c, 
bacteria;  d,  Leptothrix  buccalis  Qakob). 

especially  abundant  in  the  crypts  of  the  tonsils  and  the 
tartar  of  the  teeth.  The  whitish  patches  of  Pharyn- 
gomycosis  leptothrica  are  largely  composed  of  these  fungi. 
They  are  slender,  segmented  threads,  which  generally, 
but  not  always,  stain  violet  with  Lugol's  solution,  and  are 


378  MISCELLANEOUS   EXAMINATIONS 

readily  seen  with  a  4  mm.  objective.  At  times  they 
are  observed  in  the  sputum  and  stomach  fluid.  In  the 
former  they  might  be  mistaken  for  elastic  fibers;  in  the 
latter,  for  Boas-Oppler  bacilli.  In  either  case,  the  re- 
action with  iodin  will  distinguish  them. 

Thrush  is  a  disease  of  the  mouth  seen  most  often  in 
children,  and  characterized  by  the  presence  of  white 
patches  upon  the  mucous  membrane.  It  is  caused  by  the 
thrush  fungus,  Oidium  albicans.     When  a  bit  from  one 


Fig.  151. — Thrush  fungus  (Oidium  albicans)  (Jakob). 

of  the  patches  is  pressed  out  between  a  slide  and  cover  and 
examined  with  a  4  mm.  objective,  the  fungus  is  seen  to 
consist  of  a  network  of  branching  segmented  hyphae 
with  numerous  spores,  both  within  the  hyphae  and  in  the 
meshes  between  them  (Fig.  151).  The  meshes  also  con- 
tain leukocytes,  epithelial  cells,  and  granular  debris. 

Acute  pseudomembranous  inflammations,  which  occur 
chiefly  upon  the  tonsils  and  nasopharynx,  are  generally 
caused  by  the  diphtheria  bacillus,  but  may  result  from 


THE  MOUTH  379 

streptococcic  infection.  In  many  cases  diphtheria 
bacilli  can  be  demonstrated  in  smears  made  from  the 
membrane  and  stained  with  Loffler's  methylene-blue  or 
2  per  cent,  aqueous  solution  of  methyl-green.  They  are 
straight  or  curved  rods,  which  vary  markedly  in  size 
and  outline,  and  stain  very  irregularly.  A  characteristic 
form  is  a  palely  tinted  rod  with  several  deeply  stained 
granules    (metachromatic    bodies),    or    with    one   such 


..      •;■     -^^^  7^^^-.'  j^. 


Fig.  152. — Bacillus  diphtheriae  stained  with  methyl-green;  culture  from  throat  (  X  1000) 
(photograph  by  the  author). 

granule  at  each  end  (Fig.  152).  They  stain  by  Gram's 
method.  It  is  generally  necessary,  and  always  safer,  to 
make  a  culture  upon  blood-serum,  incubate  for  twelve 
hours,  and  examine  smears  from  the  growth. 

Vincent's  angina  is  a  pseudomembranous  and  ulcer- 
ative inflammation  of  mouth  and  pharynx,  which  when 
acute  may  be  mistaken  for  diphtheria,  and  when  chronic 
is  very  apt  to  be  mistaken  for  syphilis.     Stained  smears 


38o 


MISCELLANEOUS   EX.\MINATIONS 


from  the  ulcers  or  membrane  show  large  numbers  of 
spirochaetae  and  "  fusiform  bacilli,"  giving  a  striking  and 
characteristic  picture  (Fig.  153).  The  "bacillus"  is 
spindle  shaped,  more  or  less  pointed  at  the  ends,  and 
about  6  to  12  a  long.  The  spirillum  is  a  very  slender, 
wavy  thread,  about  30  to  40  u  long.  Diluted  analin- 
gentian-violet  makes  a  satisfactory  stain.  Further  de- 
scription is  given  on  p.  331. 


0 


^.  V, 


m  ^ 


Fig.  153. — Spirochaeta  vincenti  from  case  of  ulcerative  stomatitis  {  X  1200) 


Tuberculous  ulcerations  of  mouth  and  pharynx  can 
generally  be  diagnosed  from  curetings  made  after  careful 
cleansing  of  the  surface.  The  curetings  are  well  rubbed 
between  slide  and  cover,  and  the  smears  thus  made  are 
dried,  fixed,  and  stained  for  tubercle  bacilU.  Since  there 
is  much  danger  of  contamination  from  tuberculous  spu- 
tum, the  presence  of  tubercle  bacilli  is  significant  only  in 
proportion  to  the  thoroughness  with  which  the  ulcer  was 


THE  EYE  381 

cleansed.  The  diagnosis  is  certain  when  the  bacilli  are 
found  within  groups  of  cells  which  have  not  been  dis- 
associated in  making  the  smears. 

THE  EYE 

Staphylococci,  pneumococci,  and  streptococci  are  prob- 
ably the  most  common  of  the  bacteria  to  be  found  in  non- 
specific conjunctivitis  and  keratitis.  Serpiginous  ulcer 
of  the  cornea  is  generally  associated  with  the  pneumococ- 
cus  (Fig.  143). 


Fig.  154. — Conjunctival  secretion  from  acute  contagious  conjunctivitis;  polynuclear 
leukocytes  with  the  bacillus  of  Weeks;  P,  phagocyte  containing  bacillus  of  Weeks  (one- 
twelfth  oil-immersion,  ocular  iii)  (Morax). 

The  usual  cause  of  acute  infectious  conjunctivitis 
("pink-eye"),  especially  in  cities,  seems  to  be  the  Koch- 
Weeks  bacillus.  This  is  a  minute,  slender  rod,  which 
lies  within  and  between  the  pus-corpuscles  (Fig.  154), 
and  is  negative  to  Gram's  stain.  In  smears  it  cannot  be 
distinguished  from  the  influenza  bacillus,  although  its 
length  is  somewhat  greater. 

The  diplohacillus  of  Morax  and  Axenfeld  gives  rise 
to  an  acute  or  chronic  blepharoconjunctivitis  without 
follicles  or  membrane,  for  which  zinc  sulphate  seems  to  be 
a  specific.      It  is  widely  distributed  geographically  and 


382  MISCELLANEOUS   EXAMINATIONS 

is  common  in  many  regions.  The  organism  is  a  short, 
thick  diplobacillus,  is  frequently  intracellular,  and  is 
Gram-negative  (Fig.  155).  A  delicate  capsule  can  some- 
times be  made  out. 

Early  diagnosis  of  gonorrheal  ophthalmia  is  extremely 
important,  and  can  be  made  with  certainty  only  by  detec- 
tion of  gonococci  in  the  discharge.  They  are  easily  found 
in  smears  from  untreated  cases.     After   treatment  is 


Fig.  155. — The  diplobacillus  of  Morax  and  Axenfeld  (from  a  preparation  by  Dr.  Harold 

Gifford). 

begun  they  soon  disappear,  even  though  the  discharge 
continues. 

Pseudomembranous  conjunctivitis  generally  shows 
either  streptococci  or  diphtheria  bacilli.  In  diagnosing 
diphtheritic  conjunctivitis,  one  must  be  on  his  guard 
against  the  Bacillus  xerosis,  which  is  a  frequent  inhabit- 
ant of  the  conjunctival  sac  in  healthy  persons,  and  which 
is  identical  morphologically  with  the  diphtheria  bacillus. 


THE  EAR  383 

The  clinical  picture  is  hence  more  significant  than  the 
microscopic  findings. 

Various  micro-organisms — bacteria,  molds,  protozoa — • 
have  been  described  in  connection  with  trachoma,  but  the 
specific  organism  of  the  disease  is  not  definitely  known. 

Herbert  has  called  attention  to  the  abundance  of 
eosinophilic  leukocytes  in  the  discharge  of  vernal  catarrh. 
He  regards  their  presence  in  considerable  numbers  as 
very  helpful  in  the  diagnosis  of  this  disease. 

THE  EAR 

By  far  the  most  frequent  exciting  causes  of  acute  otitis 
media  are  the  pneumococcus  and  the  streptococcus.  The 
finding  of  other  bacteria  in  the  discharge  generally  indi- 
cates a  secondary  infection,  except  in  cases  complicating 
infectious  diseases,  such  as  typhoid  fever,  diphtheria,  and 
influenza.  Discharges  which  have  continued  for  some 
time  are  practically  always  contaminated  with  the 
staphylococcus.  The  presence  of  the  streptococcus 
should  be  a  cause  of  uneasiness,  since  it  much  more 
frequently  leads  to  mastoid  disease  and  meningitis  than 
does  the  pneumococcus.  The  staphylococcus,  bacillus 
of  Friedlander,  colon  bacillus,  and  Bacillus  pyocyaneus 
may  be  met  in  chronic  middle-ear  disease. 

In  tuberculous  disease  the  tubercle  bacillus  is  present  in 
the  discharge,  but  its  detection  offers  some  difficulties.  It 
is  rarely  easy  to  find,  and  precautions  must  always  be 
taken  to  exclude  the  smegma  and  other  acid-fast  bacilli 
(P-  53))  which  are  especially  liable  to  be  present  in  the 
ear.  Rather  striking  is  the  tendency  of  old  squamous 
cells  to  retain  the  red  stain,  and  fragments  of  such  cells 
may  mislead  the  unwary. 


384  MISCELLANEOUS   EXAMINATIONS 

PARASITIC  DISEASES  OF  THE  SKIN 

Favus,  tinea  versicolor,  and  the  various  forms  of  ring- 
worm are  caused  by  members  of  the  fungus  group.  To 
demonstrate  them,  a  crust  or  a  hair  from  the  affected  area 
is  softened  with  a  few  drops  of  20  per  cent,  caustic  soda 
solution,  pressed  out  between  a  shde  and  cover,  and 
examined  with  a  one-sixth  objective.  They  consist  of  a 
more  or  less  dense  network  of  hyphae  and  numerous 
round  or  oval  refractive  spores.  The  cuts  in  standard 
works  upon  diseases  of  the  skin  will  aid  in  differentiating 
the  members  of  the  group. 

MILK 

A  large  number  of  analyses  of  human  and  cows'  milk 
are  averaged  by  Holt  as  follows,  Jersey  milk  being  ek- 
cluded  because  of  its  excessive  fat: 

Human  Milk.  Cows'  Milk. 

Normal  variations.  Average,  per  Average,  per 

per  cent.  cent.  cent. 

Fat 3.00  to      5.CX3  4.00  3.50 

Sugar 6.00  to      7.00  7.CX3  4.30 

Proteins i.cxj  to      2.25  1.50  4.00 

Salts 0.18  to      0.25  0.20  0.70 

Water 8Q.82  to    85.50  87.30  87.50 

100.00       loo.oo  100.00  100.00 

The  reaction  of  human  milk  is  slightly  alkaline;  of 
cows',  neutral  or  slightly  acid.  The  specific  gravity  of 
each  is  about  1.028  to  1.032.  Human  milk  is  sterile  when 
secreted,  but  derives  a  few  bacteria  from  the  lacteal 
ducts.  Cows'  milk,  as  usually  sold,  contains  large  num- 
bers of  bacteria,  the  best  milk  rarely  containing  fewer 
than   10,000  per   cubic   centimeter.     Microscopically, 


MILK 


385 


human  milk  is  a  fairly  homogeneous  emulsion  of  fat, 
and  is  practically  destitute  of  cellular  elements. 

Chemic  examination  of  milk  is  of  great  value  in  solving 
the  problems  of  infant  feeding.    The  sample  examined 


r 


\ 


en 


c.c. 

0 lOl 


2_| 
3_|_  1 

4_|-6 

6_|-4 
7-|_3 
dJLz 
9_i_  I 


10_=_0 


Fig.  156. — Holt's  milk-testing  apparatus. 


should  be  the  middle  milk,  or  the  entire  quantity  from 
one  breast.  The  fat  and  protein  can  be  estimated 
roughly,  but  accurately  enough  for  many  clinical  purposes 
by  means  of  Holt's  apparatus,  which  consists  of  a  10  c.c. 
cream  gage  and  a  small  hydrometer  (Fig.  156).     The 

25 


386 


MISCELLANEOUS   EXAMINATIONS 


cream  gage  is  filled  to  the  o  mark  with  milk,  allowed  to 
stand  for  twenty-four  hours  at  room  temperature,  and  the 
percentage  of  cream  then  read  off.  The  percentage  of 
fat  is  three-fifths  that  of  the  cream.  The  protein  is  then 
approximated  from  a  consideration  of  the  specific  gravity 
and  the  percentage  of  fat.  The  salts  and  sugar  very  sel- 
dom vary  sufficiently  to  affect  the  specific  gravity,  hence 
a  high  specific  gravity  must  be  due  to  either  an  increase 
of  protein  or  decrease  of  fat,  or  both,  and  vice  versa. 
With  normal  specific  gravity  the  protein  is  high  when 
the  fat  is  high,  and  vice  versa.  The 
method  is  not  accurate  with  cows'  milk. 
For  more  accurate  work  the  following 
methods,  applicable  to  either  human  or 
cows'  milk,  are  simple  and  satisfactory. 
Fat. — Leffmann-Beam  Method.- — This 
is  essentially  the  widely  used  Babcock 
method,  modified  for  the  small  quanti- 
ties of  milk  obtainable  from  the  human 
mammary  gland.  The  apparatus  con- 
sists of  a  special  tube  which  fits  the 
aluminum  shield  of  the  medical  centri- 
fuge (Fig.  157)  and  a  5  c.c.  pipet.  Owing 
to  its  narrow  stem,  the  tube  is  difficult 
to  fill  and  to  clean.  Exactly  5  c.c.  of 
the  milk  are  introduced  into  the  tube  by  means  of  the 
pipet,  and  i  c.c.  of  a  mixture  of  equal  parts  of  concen- 
trated hydrochloric  acid  and  amyl-alcohol  is  added  and 
well  mixed.  The  tube  is  filled  to  the  o  mark  with  con- 
centrated sulphuric  acid,  adding  a  few  drops  at  a  time 
and  agitating  constantly.  This  is  revolved  in  the  centri- 
fuge at  1000  revolutions  a  minute  for  three  minutes,  or 


Fig.    iS7.^Tube    for 
milk  analysis. 


MILK  387 

until  the  fat  has  separated.  The  percentage  is  then  read 
off  upon  the  stem,  each  small  division  representing  0.2 
per  cent,  of  fat. 

Proteins. — T.  R.  Boggs'  Modification  of  the  Esbach 
Method. — This  is  applied  as  for  urinary  albumin  (p.  105), 
substituting  Boggs'  reagent  for  Esbach's.  The  reagent 
is  prepared  as  follows: 

(i)  Phosphotungstic  acid 25  gm. 

Distilled  water 125  c.c. 

(2)  Concentrated  hydrochloric  acid 25  c.c. 

Distilled  water 100  c.c. 

When  the  phosphotungstic  acid  is  completely  dissolved, 
mix  the  two  solutions.  This  reagent  is  quite  stable  if 
kept  in  a  dark  glass  bottle. 

Before  examination,  the  milk  should  be  diluted  accord- 
ing to  the  probable  amount  of  protein,  and  allowance 
made  in  the  subsequent  reading.  For  human  milk  the 
optimum  dilution  is  i  :  10;  for  cows'  milk,  i  :  20.  Dilu- 
tion must  be  accurate. 

Lactose. — The  protein  should  first  be  removed  by 
acidifying  with  acetic  acid,  boiling,  and  filtering.  Purdy's 
method  may  then  be  used  as  for  glucose  in  the  urine 
(p.  112);  but  it  must  be  borne  in  mind  that  lactose  re- 
duces copper  more  slowly  than  glucose,  and  longer  heat- 
ing is,  therefore,  required;  and  that  35  c.c.  of  Purdy's 
solution  is  equivalent  to  0.0268  gm.  lactose  (as  com- 
pared with  0.02  gm.  glucose). 

It  is  frequently  desirable  to  detect  formalin,  which 
is  the  most  common  preservative  added  to  cows'  milk. 
Add  a  few  drops  of  dilute  ferric  chlorid  solution  to  a  few 


3^^ 


MISCELLANEOUS   EXAMINATIONS 


cubic  centimeters  of  the  milk,  and  run  the  mixture 
gently  upon  the  surface  of  some  strong  sulphuric  acid 
in  a  test-tube.  If  formaldehyd  be  present,  a  bright  red 
ring  will  appear  at  the  line  of  contact  of  the  fluids.  This 
is  not  a  specific  test  for  formaldehyd,  but  nothing  else 
likely  to  be  added  to  the  milk  will  give  it. 

SYPHILITIC  MATERIAL 

In  1905  Schaudinn  and  Hoffmann  described  the 
occurrence  of  a  very  slender,  spiral  micro-organism  in 
the  lesions  of  syphilis.     This  they  named  Spirochata 


^  ^^■;^ 


Fig.  158. — Treponema  pallidum  (X  1000)  (Leitz  j'j  oil-immersion  objective  and  Lcitz 
dark-ground  condenser). 

pallida,  because  of  its  low  refractive  power  and  the 
difficulty  with  which  it  takes  up  staining  reagents.  The 
name  was  later  changed  to  Treponema  pallidum.  Its 
etiologic  relation  to  syphilis  is  now  universally  admitted. 
It  is  found  in  primary,  secondary,  and  tertiary  lesions, 
but  is  not  present  in  the  latter  in  sufficient  numbers  to 
be  of  value  in  diagnosis. 


SYPHILITIC  MATERIAL  389 

Treponema  pallidum  is  an  extremely  slender,  spiral, 
motile  thread,  with  pointed  ends.  There  is  a  flagellum 
at  each  end,  but  it  is  not  seen  in  ordinary  preparations. 
The  organism  varies  considerably  in  length,  the  average 
being  about  7  /M,  or  the  diameter  of  a  red  blood-corpuscle; 
and  it  exhibits  three  to  twelve,  sometimes  more,  spiral 
curves,  which  are  sharp  and  regular  and  resemble  the 
curves  of  a  corkscrew  (Figs.  112,  158,  159).  It  is  so 
delicate  that  it  is  difficult  to  see  even  in  well-stained 


Fig.  159. — Tr^Mnema  pallidum  and  Spiroch.'eta  refringens  (X  1200)  (Leitz  oil-immersion 

objective). 

preparations;  a  high  magnification  and  careful  focusing 
are,  therefore,  required.  Upon  ulcerated  surfaces  it  is 
often  mingled  with  other  spiral  micro-organisms,  which 
adds  to  the  difficulty  of  its  detection.  The  most  notable 
of  these  is  Spirochceta  refringens,  described  on  p.  333. 

Treponema  pallidum  is  most  easily  demonstrated  in 
chancres  and  mucous  patches,  although  the  skin  lesions 
— papules,  pustules,  roseolous  areas— often  contain  large 
numbers.  Tissue-juice  from  the  deeper  portions  of  the 
lesions  is  the  most  favorable  material  for  examination, 


390  MISCELLANEOUS   EXAMINATIONS 

because  the  organisms  are  commonly  more  abundant 
than  upon  ulcerated  surfaces  and  are  rarely  accompanied 
by  other  micro-organisms.  After  cleansing,  the  surface 
is  gently  scraped  with  a  curet  or  rubbed  briskly  with  a 
swab  of  cotton  or  gauze.  In  a  few  moments  serum  will 
exude.  The  rubbing  should  not  be  so  vigorous  as  to 
bring  the  blood,  because  the  corpuscles  may  hide  the 
treponema.  Very  thin  cover-glass  smears  are  then  made 
from  the  serum. 

Staining  Methods.— Giemsa's  stain  is  probably  the  most 
widely  used.  It  is  best  purchased  ready  prepared.  Smears 
are  fixed  in  absolute  alcohol  for  fifteen  minutes.  Ten  drops 
of  the  stain  are  added  to  lo  c.c.  of  faintly  alkaline  distilled 
water  (i  drop  of  a  i  per  cent,  solution  of  potassium  carbonate 
to  lo  c.c.  of  the  water),  and  the  fixed  smear  is  immersed  in 
this  diluted  stain  for  one  to  three  hours  or  longer.  It  is  then 
rinsed  in  distilled  water,  dried,  and  mounted.  In  well-stained 
specimens  Treponema  pallidum  is  reddish,  most  other  micro- 
organisms, bluish.  More  intense  staining  may  be  obtained 
by  gently  warming  the  stain. 

Wright's  blood-stain,  used  in  the  manner  already  described 
(p.  222)  except  that  the  diluted  stain  is  allowed  to  act  upon 
the  film  for  fifteen  minutes,  gives  good  results. 

Silver  Method.— The  silver  impregnation  method  has 
long  been  used  for  tissues.  Stein  has  applied  it  to  smears  as 
follows: 

1.  Dry  the  films  in  the  incubator  at  37°  C.  for  three  or 
four  hours. 

2.  Immerse  in  10  per  cent,  silver  nitrate  solution,  in  diffuse 
daylight  for  some  hours,  until  the  preparation  takes  on  a 
metallic  luster. 

3.  Wash  in  water,  dry,  and  mount. 

The  organisms  are  black  against  a  brownish  background. 


SEMEN  391 

India-ink  Method. — A  drop  of  India-ink  of  good  grade 
(Gunther  and  Wagner's  recommended)  is  diluted  with  i 
to  5  drops  of  water.  A  loopful  of  this  is  mixed  on  a  slide 
with  a  similar  quantity  of  serum  from  the  suspected  lesion. 
The  mixture  is  then  spread  over  the  slide  and  allowed  to  dry. 
After  drying,  it  is  examined  with  an  oil-immersion  lens. 
Micro-organisms,  including  Treponema  pallidum,  appear 
clear  white  on  a  brown  or  black  background,  much  as  they  do 
with  the  dark  ground  condenser  (Fig.  158).  Because  of  its 
extreme  simplicity  this  method  has  been  favorably  received. 
It  cannot,  however,  be  absolutely  relied  upon,  since,  as  has 
been  pointed  out,  many  India-inks  contain  wavy  vegetable 
fibrils  which  might  easily  mislead  a  beginner,  and  sometimes, 
indeed,  even  an  experienced  worker. 

Dark  groixnd  illumination  (see  p.  21)  may  be  used  to 
study  the  living  organisms  in  fresh  tissue  juices.  This  offers 
a  satisfactory  means  of  diagnosis,  but  since  the  instrument  is 
expensive  the  practitioner  will  rely  upon  one  or  more  of  the 
staining  methods  just  enumerated. 

SEMEN 

Absence  of  spermatozoa  is  a  more  common  cause  of 
sterility  than  is  generally  recognized.  In  some  cases 
they  are  present,  but  lose  their  motility  immediately 
after  ejaculation. 

Semen  must  be  kept  warm  until  examined.  When  it 
must  be  transported  any  considerable  distance,  the 
method  suggested  by  Boston  is  convenient.  The  fresh 
semen  is  placed  in  a  small  bottle,  to  the  neck  of  which  a 
string  is  attached.  This  is  then  suspended  from  a  button 
on  the  trousers,  so  that  the  bottle  rests  against  the  skin  of 
the  inguinal  region.  It  may  be  carried  in  this  way  for 
hours.     When  ready  to  examine,  place  a  small  quantity 


392 


MISCELLANEOUS   EXAMINATIONS 


upon  a  warmed  slide  and  apply  a  cover.  The  sperma- 
tozoa are  readily  seen  with  a  4  mm.  objective  (Fig.  57). 
Normally,  they  are  abundant  and  in  active  motion. 

Detection  of  semen  in  stains  upon  clothing,  etc.,  is 
often  important.  The  finding  of  spermatozoa,  after 
soaking  the  stain  for  an  hour  in  normal  salt  solution  or 


Fig.  160. — Seminal  crystals  (medium  size)  (X750)  from  a  stain  on  clothing.  A  sin- 
gle thread  i  inch  long  was  used  in  the  test,  the  stain  being  three  years  and  four 
months  old  (Peterson  and  Haines). 


dilute  alcohol,  and  teasing  in  the  same  fluid,  is  absolute 
proof  that  the  stain  in  question  is  semen,  although  it  is 
not  possible  to  distinguish  human  semen  from  that  of  the 
lower  animals  in  this  way.  A  little  eosin  added  to  the 
fluid  will  bring  the  spermatozoa  out  more  clearly. 

Florence's  Reaction. — The  suspected  material  is  soft- 
ened with  water,  placed  upon  a  slide  with  a  few  drops 


DIAGNOSIS   OF  RABIES  393 

of  the  reagent,  and  examined  at  once  with  a  medium 
power  of  the  microscope.  If  the  material  be  semen, 
there  will  be  found  dark-brown  crystals  (Fig.  i6o)  in  the 
form  of  rhombic  platelets  resembling  hemin  crystals,  or 
of  needles,  often  grouped  in  clusters.  These  crystals  can 
also  be  obtained  from  crushed  insects,  watery  extracts  of 
various  internal  organs,  and  certain  other  substances, 
so  that  they  are  not  absolute  proof  of  the  presen'ce  of 
semen.  Negative  results,  upon  the  other  hand,  are  con- 
clusive, even  when  the  semen  is  many  years  old. 

The  reagent  consists  of  iodin,  2.54  gm.;  potassium 
iodid,  1.65  gm.;  and  distilled  water,  30  c.c. 

DIAGNOSIS  OF  RABIES 

In  view  of  the  brilliant  results  attending  prophylactic 
treatment  by  the  Pasteur  method,  early  diagnosis  of 
rabies  (hydrophobia)  in  animals  which  have  bitten  per- 
sons is  extremely  important. 

The  most  reliable  means  of  diagnosis  is  the  production 
of  the  disease  in  a  rabbit  by  subdural  or  intracerebral 
injection  of  a  Httle  of  the  filtrate  from  an  emulsion  of 
the  brain  and  medulla  of  the  suspected  animal.  The 
diagnosis  can,  however,  usually  be  quickly  and  easily 
made  by  microscopic  demonstration  of  Negri  bodies. 
Whether  these  bodies  be  protozoan  in  nature  and  the 
cause  of  the  disease,  as  is  held  by  many,  or  whether  they 
be  products  of  the  disease,  it  is  certain  that  their  presence 
is  pathognomonic. 

Negri  bodies  are  sharply  outlined,  round,  oval,  or 
somewhat  irregular  structures  which  vary  in  size,  the 
extremes  being  0.5  and  18  |t/.  They  consist  of  a  hyalin- 
like  cytoplasm,  in  which  when  properly  stained  one  or 


394  MISCELLANEOUS   EXAMINATIONS 

more  chromatin  bodies  can  usually  be  seen.  They  are 
situated  chiefly  within  the  cytoplasm  of  the  large  cells 
of  the  central  nervous  system,  the  favorite  locations 
being  the  multipolar  cells  of  the  hippocampus  major 
(Ammon's  horn).  In  many  cases  they  suggest  red  blood- 
corpuscles  lying  within  nerve-cells. 

Probably  the  best  method  of  demonstrating  Negri 
bodies  is  the  impression  method  of  Langdon  Frothingham, 
which  is  carried  out  as  follows: 

(i)  Place  the  dog's  brain^  upon  a  board  about  lo  inches 
square,  and  divide  into  two  halves  by  cutting  along  the  me- 
dian line  with  scissors. 

(2)  From  one  of  the  halves  cut  away  the  cerebellum  and 
open  the  lateral  ventricle,  exposing  the  Ammon's  horn. 

(3)  Dissect  out  the  Ammon's  horn  as  cleanly  as  possible. 

(4)  Cut  out  a  small  disc  at  right  angles  to  the  long  axis  of 
the  Ammon's  horn,  so  that  it  represents  a  cross-section  of  the 
organ. 

(5)  Place  this  disc  upon  the  board  near  the  edge,  with  one 
of  the  cut  surfaces  upward. 

(6)  Press  the  surface  of  a  thoroughly  clean  slide  upon  the 
disc  and  lift  it  suddenly.  The  disc  (if  its  exposed  surface  has 
not  been  allowed  to  become  too  dry)  will  cling  to  the  board, 
leaving  only  an  impression  upon  the  slide.  Make  several 
similar  impressions  upon  different  portions  of  the  slide,  using 
somewhat  greater  pressure  each  time.  Impressions  are  also 
to  be  made  from  the  cut  surface  of  the  cerebellum,  since  Negri 
bodies  are  sometimes  present  in  the  Purkinje  cells  when  not 
found  in  the  Ammon's  horn. 

(7)  Before  the  impressions  dry,  immerse  in  methyl-alcohol 
for  one-half  to  two  minutes. 

'  For  Dr.  Frothingham's  method  of  removing  a  dog's  brain  .see  Ameri- 
can Journal  of  Public  Hygiene  for  February,  1908. 


PLATE   XIII 


V  "     fi  ^J;sr\■^»l-^ 


A , 


.--^' 


% 


Nerve-cells  containing  Negri  bodies. 

Hippocampus  impression  preparation,  dog.  Van  Gieson  stain: 
X  looo.  I,  Negri  bodies;  2,  capillary;  3,  free  red  blood-corpuscles 
(courtesy  of  Langdon  Frothingham). 


DIAGNOSIS   OF  RABIES  395 

(8)  Cover  with  Van  Gieson's  methylene-blue-fuchsin  stain, 
warming  gently  for  one-half  to  two  minutes.  This  stain,  as 
modified  by  Frothingham,  is  as  follows.  It  must  be  freshly 
mixed  each  day: 

Tap-water 20  c.c. 

Saturated  alcoholic  solution  basic  fuchsin 3  drops. 

Saturated  aqueous  solution  methylene-blue i  drop. 

(9)  Wash  in  water  and  dry  with  filter-paper.  Examine 
with  a  low  power  to  locate  the  large  cells  in  which  the 
bodies  are  apt  to  be  found,  and  study  these  with  an  oil- 
immersion  lens. 

The  Negri  bodies  are  stained  a  pale  pink  to  purplish  red, 
and  frequently  contain  small  blue  dots  (Plate  XIII).  The 
nerve-cells  are  blue,  and  red  blood-corpuscles  are  colorless 
or  yellowish-copper  colored. 

When  the  work  is  finished,  the  board  with  the  dissected 
brain  is  sterilized  in  the  steam  sterilizer. 

Demonstration  of  Negri  bodies  by  this  method  is  quicker 
and,  probably,  more  certain  than  by  the  study  of  sections. 
It  has  the  decided  advantage  over  the  smear  method  that  the 
histologic  structure  is  retained.  One  or  more  of  the  impres- 
sions generally  shows  the  entire  cell  arrangement  almost  as 
well  as  in  sections,  and  it  is  very  easy  to  locate  favorable 
fields  with  a  16  mm.  objective. 


CHAPTER  VIII 

BACTERIOLOGIC  METHODS 

Bacteriology  has  become  so  important  a  part  of  medi- 
cine that  some  knowledge  of  bacteriologic  methods  is 
imperative  for  the  present-day  practitioner.  It  has  been 
the  plan  of  this  book  to  describe  the  various  bacteria 
and  bacteriologic  methods  with  the  subjects  to  which 
they  seemed  to  be  particularly  related.  The  tubercle 
bacillus  and  its  detection,  for  example,  are  described  in 
the  chapters  upon  Sputum  and  Urine;  blood-cultures 
are  discussed  in  the  chapter  upon  Blood.  There  are, 
however,  certain  methods,  notably  the  preparation  of 
media  and  the  study  of  bacteria  by  cultures,  which  do 
not  come  within  the  scope  of  any  previous  section,  and 
an  outline  of  these  is  given  in  the  present  chapter, 

L  APPARATUS 

Much  of  the  apparatus  of  the  clinical  laboratory  is 
called  into  use.     Only  the  following  need  special  mention : 

I.  Sterilizers. — Two  are  required. 

The  dry,  or  hot-air  sterilizer,  is  a  double-walled  oven 
similar  to  the  detached  ovens  used  with  gas  and  gasolene 
stoves.  It  has  a  hole  in  the  top  for  a  perforated  cork 
with  thermometer. 

The  steam  sterilizer  is  preferably  of  the  Arnold  type, 
opening  either  at  the  top  or  the  side.     An  autoclave,  which 

39(3 


APPARATUS  397 

sterilizes  with  steam  under  pressure,  is  very  desirable, 
but  not  necessary. 

2.  Incubator. — This  is  the  most  expensive  piece  of 
apparatus  which  will  be  needed.  It  is  made  of  copper, 
and  has  usually  both  a  water-  and  an  air-jacket  surround- 
ing the  incubating  chamber.  It  is  provided  with  ther- 
mometer, thermo-regulator,  and  some  source  of  heat, 
usually  a  Koch  safety  Bunsen  burner.  With  a  little 
ingenuity  one  can  rig  up  a  drawer  or  a  small  box,  in 
which  a  fairly  constant  temperature  can  be  maintained 
by  means  of  an  electric  light.  The  degree  of  heat  can 
be  regulated  by  moving  the  drawer  in  or  out,  or  holes 
can  be  made  in  which  corks  may  be  inserted  and  removed 
as  needed.  A  Thermos  bottle  has  been  suggested  as  a 
temporary  make-shift. 

3.  Culture-tubes  and  Flasks. — For  most  work  ordinary 
test-tubes,  5  by  |  inches,  are  satisfactory.  For  special 
purposes  a  few  3  by  |  inch  and  6  by  f  inch  tubes  may 
be  needed.  Heavy  tubes,  which  do  not  easily  break,  can 
be  obtained,  and  are  especially  desirable  when  tubes  are 
cleaned  by  an  untrained  assistant. 

Flasks  of  various  sizes  are  needed.  The  Ehrlen- 
meyer  type  is  best.  Quart  and  pint  milk  bottles  and 
2-ounce  wide-mouthed  bottles  will  answer  for  most  pur- 
poses. 

4.  Platinum  Wires. — At  least  two  of  these  are  needed. 
Each  consists  of  a  piece  of  platinum  wire  about  8  cm.  long, 
fixed  in  the  end  of  a  glass  or  metal  rod.  One  is  made  of 
about  22  gage  wire  and  its  end  is  curled  into  a  loop  i  to 
2  mm.  in  diameter.  A  loop  i  mm.  in  diameter  is  some- 
times called  a  "  normal."  The  other  wire  is  somewhat 
heavier  and  its  tip  is  hammered  flat. 


398  BACTERIOLOGIC  METHODS 

5.  Pipets,  etc. — In  addition  to  the  graduated  pipets 
with  which  every  laboratory  is  suppHed,  there  are  a 
number  of  forms  which  are  generally  made  from  glass 
tubing  as  needed.  One  of  the  simplest  of  these  is  made 
as  follows:  A  section  of  glass  tubing,  about  12  cm.  long 
and  8  mm.  in  diameter,  is  grasped  at  the  ends,  and  its 
center  is  heated  in  a  concentrated  flame.  A  blast-lamp 
is  best,  but  a  Bunsen  burner  will  usually  answer,  par- 


GPOUP>   A 


Gr?OUP>    B 


Fig.  161. — Process  of  making  pipets  (group  A)  and  Wright's  capsule  (group  B).    The 
dotted  lines  indicate  where  the  glass  is  to  be  broken. 

ticularly  if  fitted  with  a  "wing"  attachment.  When  the 
glass  is  thoroughly  softened  it  is  removed  from  the  flame, 
and,  with  a  steady,  but  not  rapid  pull,  is  drawn  out  as 
shown  in  Fig.  161.  The  slender  portion  is  scratched 
near  the  middle  with  a  file  and  is  broken  to  make  two 
pipets,  which  are  then  fitted  with  rubber  nipples.  Two 
conditions  are  essential  to  success:  the  glass  must  be 
thoroughly  softened  and  it  must  be  removed  from  the 
flame  before  beginning  to  pull. 


STERILIZATION  399 

A  nipple  can  be  made  of  a  short  piece  of  rubber 
tubing,  one  end  of  which  is  plugged  with  a  glass  bead. 

This  pipet  has  many  uses  about  the  laboratory. 
When  first  made  it  is  sterile  and  may  be  used  to  transfer 
cultures.  With  a  grease-pencil  mark  about  2  cm.  from 
its  tip  (Fig.  163),  it  is  useful  for  measuring  very  small 
quantities  of  fluid,  as  in  making  dilutions  for  the  Widal 
test  and  in  counting  bacteria  in  vaccines.  Mett's  tubes 
for  pepsin  estimation  may  be  made  from  the  capillary 
portion.  The  capillary  portion  also  makes  a  very  satis- 
factory blood-lancet  if  heated  in  a  low  flame  and  drawn 
out  quickly. 

Another  useful  device  is  the  Wright  capsule,  which 

is  made  as  shown  in  Fig.  161.     Its  use  is  illustrated  in 

Fig.  ICO.      After  the  straight  end  is  sealed  the  curved 

portion  may  be  hooked  over  the  aluminum  tube  of  the 

centrifuge,  and  the  contained  blood  or  other  fluid  sedi- 

mented ;  but  the  speed  should  not  be  so  great  as  to  break 

the  capsule. 

II.  STERILIZATION 

All  apparatus  and  materials  used  in  bacteriologic 
work  must  be  sterilized  before  use. 

Glassware,  metal,  etc.,  are  heated  in  the  hot-air  steril- 
izer at  150°  to  180°  C.  for  half  an  hour.  Flasks,  bottles, 
and  tubes  are  plugged  with  cotton  before  heating. 
Petri  dishes  may  be  wrapped  in  paper  in  sets  of  three. 
Pipets  and  glass  and  metal  h)^odermic  syringes  are 
placed  in  cotton-stoppered  test-tubes. 

Culture-media  and  other  fluids  must  be  sterilized  by 
steam.  Exposure  in  an  autoclave  to  a  temperature  of 
110°  C.  (6  pounds  pressure)  for  one-half  hour  is  sufficient. 
With  the  Arnold  sterilizer,  which  is  more  commonly 


4CX)  BACTERIOLOGIC  METHODS 

used,  the  intermittent  plan  must  be  adopted,  since 
steam  at  ordinary  pressure  will  not  kill  spores.  This  con- 
sists in  steaming  for  thirty  to  forty-five  minutes  on  three 
or  four  successive  days.  Spores  which  are  not  destroyed 
upon  the  first  day  develop  into  the  vegetative  form  and 
are  destroyed  at  the  next  heating.  Gelatin  media  must 
not  be  exposed  to  steam  for  more  than  twenty  minutes 
at  a  time,  and  must  then  be  removed  from  the  sterilizer 
and  cooled  in  cold  water,  otherwise  the  gelatin  may  lose 
its  power  to  solidify. 

Cotton  and  gauze  are  sterilized  by  either  hot  air  or 
steam,  preferably  the  latter. 

III.  PREPARATION  OF  CULTURE-TUBES 

New  tubes  should  be  washed  in  a  very  dilute  solution 
of  nitric  acid,  rinsed  in  clear  water,  and  allowed  to  drain 
dry. 

Tubes  which  contain  dried  culture-media  are  cleaned 
with  a  test-tube  brush  after  boiling  in  a  strong  solution  of 
washing-soda.  They  are  then  rinsed  successively  in 
clear  water,  acidulated  water,  and  clear  water,  and  al- 
lowed to  drain. 

The  tubes  are  now  ready  to  be  plugged  with  raw  cotton 
— the  "cotton  batting"  of  the  dry  goods  stores.  This  is 
done  by  pushing  a  wad  of  cotton  into  each  tube  to  a 
depth  of  about  3  cm.  with  a  glass  rod.  The  plugs  should 
fit  snugly,  but  not  too  tightly,  and  should  project  from 
the  tube  sufficiently  to  be  readily  grasped  by  the  fingers. 
The  tubes  are  next  placed  in  wire  baskets  and  heated  in 
an  oven  for  about  one-half  hour  at  150°  C.  in  order  to 
mold  the  stoppers  to  the  shape  of  the  tubes.  The  heat- 
ing should  not  char  the  cotton,  although  a  sHght  brown- 


CULTURE-MEDIA   r  n  r~  40I 

ing  does  no  harm.    The  tub^s  are  noW^sta-i^Y  ^f^9  filled 
with  culture-media.  ''  '^-((^(Al,(r  r, '^^,    ^^- ' // fC 

IV.  CULTURE-MEDIA 

For  a  careful  study  of  bacteria  a  great  variety  of  culture- 
media  is  required,  but  only  a  few — bouillon,  agar  or  solid- 
ified blood-serum,  and  gelatin — are  much  used  in  routine 
work. 

Preparation  of  Culture-media. — 

Beef  Infusion 

Hamburger  steak,  lean 500  grams. 

Tap-water icxdo  c.c. 

Mix  well;  let  soak  about  twenty-four  hours  in  an  ice- 
chest,  and  squeeze  through  cheese-cloth.  This  infusion 
is  not  used  by  itself,  but  forms  the  basis  for  various 
media.  *'  Double  strength  "  infusion,  used  in  making 
agar-agar,  requires  equal  parts  of  the  meat  and  water. 

Infusion  Bouillon 

Beef  infusion 1000  c.c. 

Peptone  (Witte) 10  grams. 

Salt 5      " 

Boil  until  dissolved;  bring  to  original  bulk  with 
water;  adjust  reaction;  and  filter. 

Beef  Extract  Bouillon 

Liebig's  extract  of  beef 3  grams. 

Peptone 10      " 

Salt 5      " 

Tap-water 1000  c.c. 

When  all  ingredients  are  dissolved,  cool,  and  beat 
in  the  whites  of  two  eggs;  boil  briskly  for  five  minutes 
and  filter.  It  is  not  usually  necessary  to  adjust  the  re- 
action. 

26 


402  ^CTERIOLOGIC  METHODS 

■  ,  ■    o      A*-''    "  '       Agak-agar 

Preparation  of  this  medium  usually  gives  the  student 
much  trouble.  There  should  be  no  difficulty  if  the  direc- 
tions are  carefully  carried  out. 

Agar-agar,  powdered  or  in  shreds 15  grams. 

Tap- water • 500  c.c. 

Boil  until  thoroughly  dissolved  and  add — 

Peptone 10  grams 

Salt S      " 

When  these  have  dissolved,  replace  the  water  lost  in 
boiling,  cool  to  about  60°  C,  and  add  500  c.c.  double- 
strength  beef  infusion.  Bring  slowly  to  the  boil,  adjust- 
ing the  reaction  meanwhile,  and  boil  for  at  least  five 
minutes.  Filter  while  hot  through  a  moderately  thick 
layer  of  absorbent  cotton  wet  with  hot  water  in  a  hot 
funnel.  A  piece  of  coarse  wire  gauze  should  be  placed 
in  the  funnel  underneath  the  cotton  to  give  a  larger  filter- 
ing surface.  This  medium  will  be  clear  enough  for  or- 
dinary work.  If  an  especially  clear  agar  is  desired,  it 
can  be  filtered  through  paper  in  an  Arnold  sterilizer. 

Agar  can  also  be  made  by  boiling  15  grams  of  powdered 
agar  in  1000  c.c.  of  bouillon  until  dissolved,  replacing 
the  water  lost  in  boiling,  and  filtering  through  paper 
in  a  sterilizer.     It  can  be  cleared  with  egg  if  desired. 

Glycerin  Agar-agar 
To  1000  c.c.  melted  agar  add  60  to  70  c.c.  glycerin. 

Gelatin 
Dissolve  100  to  120  grams  "golden  seal"  gelatin  in 
1000  c.c.  nutrient  bouillon  with  as  little  heat  as  possible, 


CULTURE-MEDIA  403 

adjust  the  reaction,  cool,  beat  in  the  whites  of  two  eggs, 
boil,  and  filter  hot  through  filter-paper  wet  with  hot 
water.  Sterilize  in  an  Arnold  sterilizer  for  twenty  min- 
utes upon  three  successive  days  and  cool  in  cold  water 
after  each  heating. 

Sugar  Media 
Any  desired  sugar  may  be  added  to  bouillon,  agar,  or 
gelatin  in  proportion  of  10  grams  to  the  liter.  Dextrose 
is  most  frequently  required.  When  other  sugars  are 
added,  media  made  from  beef-extract  should  be  used, 
since  those  made  from  beef-infusion  contain  enough  dex- 
trose to  cause  confusion. 

Loffler's  Blood-serum 

I  per  cent,  dextrose-bouillon i  part. 

Blood-serum 3  parts. 

Mix  and  tube.  Place  in  an  inspissator  at  the  proper 
slant  for  three  to  six  hours  at  80°  to  90°  C.  When  firmly 
coagulated,  steriUze  in  the  usual  way,  A  large  "double- 
cooker"  makes  a  satisfactory  inspissator.  The  tubes 
are  placed  in  the  inner  compartment  at  the  proper  slant, 
a  lid  with  perforation  for  a  thermometer  is  apphed,  and 
the  whole  is  weighted  down  in  the  water  of  the  outer 
compartment. 

Blood-serum  is  obtained  as  follows:  Beef  or  pig  blood 
is  collected  in  a  bucket  at  the  slaughter-house  and 
placed  in  an  ice-chest  until  coagulated.  The  clot  is  then 
gently  loosened  from  the  wall  of  the  vessel.  After  about 
twenty-four  hours  the  serum  will  have  separated  nicely 
and  can  be  siphoned  off.  It  is  then  stored  in  bottles 
with  a  little  chloroform  until  needed.  Red  cells,  if 
abundant,  darken  the  medium,  but  do  no  harm. 


404  BACTERIOLOGIC  METHODS 

Solidified  blood-serum  is  probably  the  most  satisfac- 
tory medium  for  general  purposes.  Nearly  all  patho- 
genic organisms  grow  well  upon  it. 

Hemoglobin  Medium 

The  simplest  way  to  prepare  this  is  to  smear  a  drop 
of  blood,  obtained  by  puncture  of  the  finger,  over  the 
surface  of  an  agar-slant,  and  to  incubate  over  night  to 
make  sure  of  sterility.  It  is  used  for  growing  the  influ- 
enza bacillus. 

Litmus  Milk 

Fresh  milk  is  steamed  in  an  Arnold  sterilizer  for  half 
an  hour,  and  placed  in  the  ice-chest  over  night.  The 
milk  is  siphoned  off  from  beneath  the  cream,  and  sufl&- 
cient  aqueous  solution  of  litmus  or,  preferably,  azolitmin 
•  is  added  to  give  a  blue- violet  color.  It  is  then  tubed 
and  sterilized. 

Potato 

Cylinders  about  one-half  inch  thick  are  cut  from 
potato  and  spHt  obHquely.  These  are  soaked  over 
night  in  running  water  and  placed  in  large  tubes,  in  the 
bottom  of  which  is  placed  a  little  cotton  saturated  with 
water.  They  are  sterilized  for  somewhat  longer  periods 
than  ordinary  media. 

Dunham's  Peptone  Solution 

Peptone lo  grams. 

Salt 5   " 

Water looo  c.c. 

Dissolve  by  boiling;  filter,  "tube,  and  sterilize. 
This  medium  is  used  to  determine  indol  production. 
To   a   twenty-four-   to   forty-eight-hour-old   culture  is 


CULTURE-MEDIA  405 

added  5  to  10  drops  of  concentrated  c.  p.  sulphuric  acid 
and  I  c.c.  of  i  :  10,000  solution  of  sodium  nitrite.  Ap- 
pearance of  a  pink  color  shows  the  presence  of  indol. 
A  pink  color  before  the  nitrite  is  added  shows  the  presence 
of  both  indol  and  nitrites. 

Hiss'  Serum  Media 

Blood-serum i  part. 

Water 3  parts. 

Warm  and  adjust  reaction  to  +  0.2  to  +  0.8.  Add 
Utmus  or  azolitmin  solution  to  give  a  blue-violet  color. 
Finally,  add  i  per  cent,  of  inulin  or  any  desired  sugar. 
The  inulin  medium  is  very  useful  in  distinguishing  be- 
tween the  pneumococcus  and  streptococcus. 

Bile  Medium 

Ox-  or  pig-bile  is  obtained  at  the  slaughter-house, 
tubed,  and  sterilized.  This  is  used  especially  for  grow- 
ing typhoid  bacilli  from  the  blood  during  Ufe.  The  fol- 
lowing is  probably  as  satisfactory  as  fresh  bile  and  is 
more  convenient: 

Inspissated  ox-bile   (Merck) 30.0  grams. 

Peptone 2.5 

Water 250.0  c.c. 

Dissolve,  place  in  tubes,  and  sterilize. 

Reaction  of  Media. — The  chemic  reaction  of  the 
medium  exerts  a  marked  influence  upon  the  growth  of 
bacteria.  It  is  adjusted  after  all  ingredients  are  dis- 
solved by  adding  sufficient  caustic  soda  solution  to 
overcome  the  acidity  of  the  meat  and  other  substances 
used.  In  general,  the  most  favorable  reaction  lies 
between  the  neutral  points  of  Utmus  and  phenolphtha- 


4o6  BACTERIOLOGIC  METHODS 

lein,  representing  a  very  faint  alkalinity  to  litmus. 
In  routine  work  it  is  usually  sufficient  to  test  with 
litmus-paper.  When  greater  accuracy  is  demanded, 
the  following  method  should  be  used:  After  all  ingre- 
dients are  dissolved  and  the  loss  during  boiling  has 
been  replaced  with  water,  lo  c.c.  of  the  medium  are 
transferred  to  an  evaporating  dish,  diluted  with  40  c.c. 
of  water,  and  boiled  for  three  minutes  to  drive  off  carbon 
dioxid.  One  c.c.  of  0.5  per  cent.  alcohoUc  solution  of 
phenolphthalein  is  then  added,  and  decinormal  sodium 
hydroxid  solution  is  run  in  from  a  buret  until  the  neutral 
point  is  reached,  indicated  by  the  appearance  of  a  per- 
manent pink  color.  The  number  of  cubic  centimeters  of 
decinormal  solution  required  to  bring  this  color  indicates 
the  number  of  cubic  centimeters  of  normal  sodium  hy- 
droxid solution  which  will  be  required  to  neutralize  100 
c.c.  of  the  medium .  The  standard  reaction  is  -I- 1 .5,  which 
means  that  the  medium  must  be  of  such  degree  of  acidity 
that  1.5  c.c.  of  normal  solution  would  be  required  to 
neutralize  100  c.c.  This  corresponds  to  faint  alkalinity 
to  litmus.  Most  pathogenic  bacteria  grow  better  with  a 
reaction  of  +1.0  or  -I- 0.8.  Example:  If  the  10  c.c.  which 
were  titrated  required  2  c.c.  of  decinormal  solution  to 
bring  the  pink  color,  the  reaction  is  +2;  and  0.5  c.c.  of 
normal  sodium  hydroxid  must  be  added  to  each  100  c.c. 
of  the  medium  to  reduce  it  to  the  standard,  +1.5. 

Tubing  Culture-media. — The  finished  product  is 
stored  in  flasks  or  distributed  into  test-tubes.  This  is 
done  by  means  of  a  funnel  fitted  with  a  section  of  rubber 
tubing  with  a  glass  tip  and  a  pinch-cock.  Great  care 
must  be  exercised,  particularly  with  media  which  solid- 
ify, not  to  smear  any  of  it  upon  the  inside  of  the  mouth  of 


STAINING  METHODS  407 

the  tube,  otherwise  the  cotton  stopper  will  stick.  Tubes 
are  generally  filled  to  a  depth  of  3  or  4  cm.  For  stab- 
cultures  a  greater  depth  is  desired. 

After  tubing,  all  culture-media  must  be  sterilized  as 
already  described.  Agar-tubes  are  cooled  in  a  slanting 
position  to  secure  the  proper  surface  for  inoculation. 

Media  should  be  stored  in  a  cool  place,  preferably  an 
ice-chest.  Evaporation  may  be  prevented  by  covering 
the  tops  of  the  tubes  with  tin-foil  or  the  rubber  caps 
which  are  sold  for  the  purpose;  or  the  cotton  stopper 
may  be  pushed  in  a  short  distance  and  a  cork  inserted. 

V.  STAINING  METHODS 

In  general,  bacteria  are  stained  to  determine  their 
morphology,  their  reaction  with  special  methods  (e.  g., 
Gram's  method),  and  the  presence  or  absence  of  certain 
structures,  as  spores,  flagella,  and  capsules.  Staining 
methods  for  various  purposes  and  the  formulae  of  the 
staining  fluids  have  been  given  in  previous  chapters  and 
can  be  found  by  consulting  the  index.  The  following 
will  probably  be  most  frequently  used: 

Methods  for  tubercle  bacilli,  pp.  49,  51,  and  168. 

Methods  for  capsules  of  bacteria,  pp.  55  and  368. 

Methods  for  Treponema  pallidum,  p.  390. 

Loffler's  alkaline  methylene-blue,  p.  57. 

Blood-stains,  pp.  221-224. 

-  The  method  of  staining  bacteria  for  morphology  is  as 
follows,  using  any  simple  bacterial  stain: 

(i)  Make  a  thin  smear  upon  a  slide  or  cover-glass. 

(2)  Dry  in  the  air,  or  by  warming  high  above  the  flame. 


4o8  BACTERIOLOGIC  METHODS 

(3)  "  Fix  "  bypassing  the  preparation,  film  side  up,  rather 
slowly  through  the  flame  of  a  Bunsen  burner:  a  cover-glass 
three  times,  a  slide  about  twelve  times.  Take  care  not  to 
scorch. 

(4)  Apply  the  stain  for  the  necessary  length  of  time,  gen- 
erally one-quarter  to  one  minute. 

(5)  Wash  in  water. 

(6)  Dry  by  waving  high  above  a  flame  or  by  blotting  with 
filter-paper. 

(7)  Mount  by  pressing  the  cover,  film  side  down,  upon  a 
drop  of  Canada  balsam  on  a  slide.  Slides  may  be  examined 
with  the  oil-immersion  lens  without  a  cover-glass. 

Simple  Bacterial  Stains. — A  simple  solution  of  any 
basic  anilin  dye  (methylene-blue,  basic  fuchsin,  gentian 
violet,  etc.)  will  stain  nearly  all  bacteria.  These 
simple  solutions  are  not  much  used  in  the  clinical 
laboratory,  because  other  stains,  such  as  Loffler's 
methylene-blue  and  Pappenheim's  pyronin-methyl- 
green  stain,  which  serve  the  purpose  even  better,  are 
at  hand. 

Pappenheim's  Pyronin-methyl-green  Stain. — This  so- 
lution colors  bacteria  red  and  nuclei  of  cells  blue.  It 
is,  therefore,  especially  useful  for  intracellular  bacteria 
like  the  gonococcus  and  the  influenza  bacillus.  It  is  a 
good  stain  for  routine  purposes,  and  is  a  most  excellent 
contrast  stain  for  Gram's  method.  It  colors  the  cyto- 
plasm of  lymphocytes  bright  red,  and  has  been  used  as  a 
differential  stain  for  these  cells.  The  solution  is  applied 
cold  for  one-half  to  five  minutes.  It  consists  of  saturated 
aqueous  solution  methyl-green,  3  to  4  parts,  and  saturated 
aqueous  solution  pyronin,  i  to  i|  parts. 

Carbol  Thionin. — Saturated  solution  thionin  in  50  per 


STAINING  METHODS  409 

cent,  alcohol,  20  c.c;  2  per  cent,  aqueous  solution  phenol, 
icx)  c.c. 

This  is  especially  useful  in  counting  bacteria  for  a 
vaccine  (p.  422).  It  can  be  used  as  a  general  stain.  In 
blood  work  it  is  used  for  the  malarial  parasite  and 
for  demonstration  of  basophilic  degeneration  of  the 
red  cells.  It  should  be  preceded  by  fixation  for  about 
a  minute  in  saturated  aqueous  solution  of  mercuric 
chlorid. 

Gram's  Method. — This  is  a  very  useful  aid  in  differen- 
tiating certain  bacteria  and  should  be  frequently  resorted 
to.  It  depends  upon  the  fact  that  when  treated  succes- 
sively with  gentian-violet  and  iodin,  certain  bacteria 
(owing  to  formation  of  insoluble  compounds)  retain  the 
stain  when  treated  with  alcohol,  whereas  others  quickly 
lose  it.  The  former  are  called  Gram-positive,  the  latter, 
Gram-negative.  In  order  to  render  Gram-negative  or- 
ganisms visible,  some  contrasting  counter  stain  is  com- 
monly applied,  but  this  is  not  a  part  of  Gram's  method 
proper. 

(i)  Make  smears,  dry,  and  fix  by  heat. 

(2)  Apply  anilin-gentian-violet  or  formalin-gentian-violet 
(p.  57)  two  to  five  minutes. 

(3)  Wash  with  water. 

(4)  Apply  Gram's  iodin  solution  one-half  to  two  minutes. 

(5)  Wash  in  alcohol  until  the  purple  color  ceases  to  come 
off.  This  is  conveniently  done  in  a  watch-glass.  The  prep- 
aration is  placed  in  the  alcohol,  face  downward,  and  one 
edge  is  raised  and  lowered  with  a  needle.  As  long  as  any 
color  is  coming  off,  purple  streaks  will  be  seen  diffusing  into 
the  alcohol  where  the  surface  of  the  fluid  meets  the  smear. 
If  forceps  be  used,  beware  of  stain  which  may  have  dried 


4IO  BACTERIOLOGIC  METHODS 

upon  them.     The  thinner  portions  of  smears  from  pus  should 
be  practically  colorless  at  this  stage. 

(6)  Apply  a  contrast  stain  for  one-half  to  one  minute. 
The  stains  commonly  used  for  this  purpose  are  an  aqueous 
or  alcoholic  solution  of  Bismarck  brown  and  a  weak  solution 
of  fuchsin.  In  the  writer's  experience,  Pappenheim's  pyro- 
nin-methyl-green  mixture  is  much  more  satisfactory;  it 
brings  out  Gram-negative  bacteria  more  sharply,  and  is 
especially  desirable  for  intracellular  Gram-negative  organ- 
isms like  the  gonococcus  and  influenza  bacillus,  since  the 
bacteria  are  bright  red  and  nuclei  of  cells  blue. 

(7)  Wash  in  water,  dry,  and  mount  in  balsam. 

The  more  important  bacteria  react  to  this  staining 
method  as  follows: 

Gram  Staining  Gram  Decolorizing 

(Deep  purple).  (Colorless  unless  a  counterstain  is  used). 

Staphylococcus.  Gonococcus. 

Streptococcus.  Meningococcus. 

Pneumococcus.  Micrococcus  catarrhalis. 

Bacillus  diphtheriae.  Bacillus  of  influenza. 

Bacillus  tuberculosis.  Typhoid  bacillus. 

Bacillus  of  anthrax.  Bacillus  coli  communis. 

Bacillus  of  tetanus.  Spirillum  of  Asiatic  cholera. 

Bacillus  aerogenes  capsulatus.  Bacillus  pyocyaneus. 

Bacillus  of  Friedlander. 

Koch-Weeks  bacillus. 

Bacillus  of  Morax-Axenfeld. 

Moeller's  Method  for  Spores. — Bodies  of  bacteria  are 
blue,  spores  are  red. 

(i)  Make  thin  smears,  dry,  and  fix. 

(2)  Wash  in  chloroform  for  two  minutes. 

(3)  Wash  in  water. 

(4)  Apply  5  per  cent,  solution  of  chromic  acid  one-half  to 
two  minutes. 


STAINING   METHODS  4II 

(5)  Wash  in  water. 

(6)  Apply  carbol-fuchsin  and  heat  to  boiling. 

(7)  Decolorize  in  5  per  cent,  solution  of  sulphuric  acid. 

(8)  Wash  in  water. 

(9)  Apply  I  per  cent,  aqueous  solution  of  methylene-blue 
one-half  minute. 

(10)  Wash  in  water,  dry,  and  mount. 

Loffler's  Method  for  Flagella. — The  methods  for 
flagella  are  applicable  only  to  cultures.  Enough  of  the 
growth  from  an  agar-culture  (which  should  not  be  more 
than  eighteen  to  twenty-four  hours  old)  to  produce  faint 
cloudiness  is  added  to  distilled  water.  A  small  drop  of 
this  is  placed  on  a  cover-glass,  spread  by  tilting,  and  dried 
quickly.  The  covers  must  be  absolutely  free  from  grease. 
To  insure  this,  they  may  be  warmed  in  concentrated 
sulphuric  acid,  washed  in  water,  and  kept  in  a  mixture 
of  alcohol  and  strong  ammonia.  When  used  they  are 
dried  upon  a  fat-free  cloth. 

(i)  Fix  by  heating  the  cover  over  a  flame  while  holding 
in  the  fingers. 

(2)  Cover  with  freshly  filtered  mordant  and  gently  warm 
for  about  a  minute. 

The  mordant  consists  of: 

20  per  cent,  aqueous  solution  of  tannic  acid 10  c.c. 

Saturated  solution  ferrous  sulphate,  cold 5  c.c. 

Saturated  aqueous  or  alcoholic  solution  gentian  violet . .     i  c.c. 

(3)  Wash  in  water. 

(4)  Apply  freshly  filtered  anilin-gentian-violet,  warming 
gently  for  one-half  to  one  minute. 

(5)  Wash  in  water,  dry,  and  mount  in  balsam. 


412  BACTERIOLOGIC  METHODS 

VI.  METHODS  OF  STUDYING  BACTERIA 

The  purpose  of  bacteriologic  examinations  is  to  de- 
termine whether  bacteria  are  present  or  not,  and,  if 
present,  their  species  and  comparative  numbers.  In 
general,  this  is  accomplished  by:  i,  Direct  microscopic 
examination;  2,  Cultural  methods;  3,  Animal  inocu- 
lation. 

1.  Microscopic  Examination. — Every  bacteriologic 
examination  should  begin  with  a  microscopic  study  of 
smears  from  the  pathologic  material,  stained  with  a  gen- 
eral stain,  by  Gram's  method,  and  often  by  the  method 
for  the  tubercle  bacillus.  This  yields  a  great  deal  of 
information  to  the  experienced  w^orker,  and  in  many  cases 
is  all  that  is  necessar}-  for  the  purpose  in  view.  It  will 
at  least  give  a  general  idea  of  what  is  to  be  expected,  and 
may  determine  future  procedure. 

2.  Cultural  Methods.— (i)  Collection  of  Material. — 
Material  for  examination  must  be  collected  under  abso- 
lutely aseptic  conditions.  It  may  be  obtained  with  a 
platinum  wire — which  has  been  heated  to  redness  just 
previously  and  allowed  to  cool — or  with  a  swab  of  sterile 
cotton  on  a  stifif  wire  or  wooden  applicator.  Such  swabs 
may  be  placed  in  cotton-stoppered  test-bubes,  sterilized, 
and  kept  on  hand  ready  for  use.  Fluids  which  contain 
very  few  bacteria,  and  hence  require  that  a  consider- 
able quantity  be  used,  may  be  collected  in  a  sterile 
hypodermic  syringe  or  one  of  the  pipets  described  on 
p.  398.  The  method  of  obtaining  blood  for  cultures 
is  given  on  p.  245. 

(2)  Inoculating  Media. — The  material  is  thoroughly 
distributed  over  the  surface  of  some  solid  medium,  solid- 


METHODS   or  STUDYING  BACTERIA  413 

ified  blood-serum  being  probably  the  best  for  routine  work. 
When  previous  examination  of  smears  has  shown  that 
many  bacteria  are  to  be  expected,  a  second  tube  should 
be  inoculated  from  the  first,  and  a  third  from  the  second, 
so  as  to  obtain  isolated  colonies  in  at  least  one  of  the  tubes. 
The  platinum  wire  must  be  heated  to  redness  before  and 
after  each  inoculation.  When  only  a  few  organisms  of 
a  single  species  are  expected,  as  is  the  case  in  blood-cul- 
tures, a  considerable  quantity  of  the  material  is  mixed 
with  a  fluid  medium. 

(3)  Incubation. — Cultures  are  placed  in  an  incubator 
which  maintains  a  uniform  temperature,  usually  of  37.5° 
C,  for  eighteen  to  twenty-four  hours,  and  the  growth,  if 
any,  is  studied  as  described  later.  Gelatin  will  melt  with 
this  degree  of  heat,  and  must  be  incubated  at  about 
room-temperature. 

(4)  Study  of  Cultures. — ^When  the  original  culture 
contains  more  than  one  species,  they  must  be  separated, 
or  obtained  in  "pure  culture,"  before  they  can  be  studied 
satisfactorily.  To  accompHsh  this  it  is  necessary  to  so 
distribute  them  on  solid  media  that  they  form  separate 
colonies,  and  to  inoculate  fresh  tubes  from  the  individual 
colonies.  In  routine  work  the  organisms  can  be  suffi- 
ciently distributed  by  drawing  the  infected  wire  over  the 
surface  of  the  medium  in  a  series  of  streaks.  If  a  suffi- 
cient number  of  streaks  be  made,  some  of  them  are 
sure  to  show  isolated  colonies.  Another  method  of 
obtaining  isolated  colonies  is  to  inoculate  the  water 
of  condensation  of  a  series  of  tubes,  the  first  from 
the  second,  the  second  from  the  third,  etc.,  and,  by 
tilting,  to  flow  the  water  once  over  the  surface  of  the 
medium. 


414  BACTERIOLOGIC  METHODS 

In  order  to  determine  the  species  to  which  an  organism 
belongs  it  is  necessary  to  consider  some  or  all  of  the  fol- 
lowing points: 

(i)  Naked-eye  and  microscopic  appearance  of  the  col- 
onies on  various  media. 

(2)  Comparative  luxuriance  of  growth  upon  various 
media.  The  influenza  bacillus,  for  example,  can  be 
grown  upon  media  containing  hemoglobin,  but  not  on 
the  ordinary  media. 

(3)  Morphology,  special  staining  reactions,  and  the 
presence  or  absence  of  spores,  flagella,  and  capsules. 
Staining  methods  for  these  purposes  have  been  given. 

(4)  Motility.  This  is  determined  by  observing  the 
living  organism  with  an  oil-immersion  lens  in  a  hanging- 
drop  preparation,  made  as  follows:  A  small  drop  of  a 
bouillon  culture  or  of  water  of  condensation  from  an 
agar  or  blood-serum  tube  is  placed  upon  the  center  of 
a  cover-glass;  this  is  inverted  over  the  concavity  of  a 
"  hollow  slide,"  and  ringed  with  vaselin.  In  focusing, 
the  edge  of  the  drop  should  be  brought  into  the  field. 
Great  care  must  be  exercised  not  to  break  the  cover  by 
pushing  the  objective  against  it.  A  method  which  in 
some  respects  is  preferable  to  the  hanging  drop  is  to 
make  a  ring  of  vaselin  on  a  slide,  place  a  drop  of  the 
culture  in  this,  and  apply  a  cover. 

It  is  not  always  easy  to  determine  whether  an  organism 
is  or  is  not  motile,  since  the  motion  of  currents  and  the 
Brownian  motion  which  affects  all  particles  in  suspen- 
sion are  sometimes  very  deceptive. 

(5)  Production  of  chemic  changes  in  the  media. 
Among  these  are  coagulation  of  milk;  production  of  acid 
in  milk  and  various  sugar  media  to  which  litmus  has  been 


CHARACTERISTICS   OF  SPECIAL   BACTERIA  415 

added  to  detect  the  change;  production  of  gas  in  sugar 
media,  the  bacteria  being  grown  in  fermentation  tubes 
similar  to  those  used  for  sugar  tests  in  urine;  and  pro- 
duction of  indol. 

(6)  Ability  to  grow  without  oxygen.  For  anaerobic 
methods,  the  reader  is  referred  to  larger  works. 

(7)  Effects  produced  when  inoculated  into  animals. 
3.  Animal    Inoculation. — In  clinical  work  this  is 

resorted  to  chiefly  to  detect  the  tubercle  bacillus.  The 
method  is  described  on  p.  375. 

For  the  study  of  bacteria  in  cultures,  a  small  amount 
of  a  pure  culture  is  injected  subcutaneously  or  into  the 
peritoneal  cavity.  The  animals  most  used  are  guinea- 
pigs,  rabbits,  and  mice.  For  intravenous  injection  the 
rabbit  is  used  because  of  the  easily  accessible  marginal 
vein  of  the  ear. 

VII.  CHARACTERISTICS  OF  SPECIAL  BACTERIA 

Owing  to  the  great  number  of  bacterial  species,  most 
of  which  have  not  been  adequately  studied,  positive 
identification  of  an  unknown  organism  is  often  a  very 
difficult  problem.  Fortunately,  however,  only  a  few 
are  commonly  encountered  in  routine  work,  and  identi- 
fication of  these  with  comparative  certainty  presents  no 
great  difficulty.  Their  more  distinctive  characteristics 
are  outlined  in  this  section. 

I .  Staphylococcus  Pyogenes  Aureus.— The  morphology 
and  staining  reactions  (described  on  p.  368)  and  the  ap- 
pearance of  the  colonies  are  sufficient  for  diagnosis. 
Colonies  on  solidified  blood-serum  and  agar  are  rounded, 
slightly  elevated,  smooth  and  shining,  and  vary  in  color 


4l6  BACTERIOLOGIC  METHODS 

from  light  yellow  to  deep  orange.     Young  colonies  are 
sometimes  white. 

2.  Staphylococcus  Pyogenes  Albus. — This  is  similar 
to  the  above,  but  colonies  are  white.  It  is  generally 
less  virulent. 

3.  Staphylococcus  Pyogenes  Citreus. — The  colonies 
are  lemon  yellow;  otherwise  it  resembles  the  white 
staphylococcus. 

4.  Streptococcus  F*yogenes. — The  morphology  and 
staining  reactions  have  been  described  (p.  368).  The 
chains  are  best  seen  in  the  water  of  condensation  and  in 
bouillon  cultures.  The  cocci  are  not  motile.  Colonies 
on  blood-serum  are  minute,  round,  grayish,  and  trans- 
lucent. Litmus  milk  is  usually  acidified  and  coagulated, 
although  slowly.  The  streptococcus  rarely  produces 
acid  in  Hiss'  serum-water-litmus-inulin  medium  (see 
p.  405). 

5.  Pneumococcus. — The  only  organism  with  which  this 
is  likely  to  be  confused  is  the  streptococcus.  The  dis- 
tinction is  often  extremely  difficult. 

Detection  of  the  pneumococcus  in  fresh  material  has 
been  described  (p.  54).  In  cultures  it  frequently  forms 
long  chains.  Capsules  are  not  present  in  cultures  ex- 
cept upon  special  media.  They  show  best  upon  a 
serum  medium  like  that  described  for  the  gonococcus, 
but  can  frequently  be  seen  in  milk.  Colonies  on  blood- 
serum  resemble  those  of  the  streptococcus.  The  pneumo- 
coccus usually  promptly  acidifies  and  coagulates  milk, 
and  acidifies  and  coagulates  Hiss'  serum-water  with 
inulin.  The  latter  property  is  very  helpful  in  diag- 
nosis. 

6.  Gonococcus. — Its  morphology  and  staining  pecu- 


CHARACTERISTICS   OF   SPECIAL  BACTERIA  417 

liarities  are  given  on  p.  369.  These  usually  suffice  for 
its  identification,  cultural  methods  being  rarely  under- 
taken. In  cultures  the  chief  diagnostic  point  is  its  failure 
to  grow  on  ordinary  media.  To  grow  it  the  most  con- 
venient mediimi  is  made  by  adding  ascitic  or  hydrocele 
fluid  (which  has  been  obtained  under  aseptic  conditions) 
to  melted  agar  in  proportion  of  i  part  of  serum  to 
3  parts  of  agar.  The  agar  in  tubes  is  melted  and 
cooled  to  about  45°  C;  the  serum  is  added  with  a  pipet 
and  mixed  by  shaking;  and  the  tubes  are  again  cooled  in 
a  slanting  position.  Colonies  upon  this  medium  are 
minute,  grayish,  and  translucent. 

7.  Diplococcus  Intracellularis  Meningitidis. — It  grows 
poorly  or  not  at  all  on  plain  agar.  On  Loffler's  blood- 
serum,  upon  which  it  grows  fairly  well,  colonies  are  round, 
colorless  or  hazy,  flat,  shining,  and  viscid  looking.  It 
quickly  dies  out. 

8.  Diphtheria  Bacillus. — The  diagnosis  is  usually 
made  from  a  study  of  stained  smears  from  cultures 
upon  blood-serum.  Its  morphology  and  staining  pecu- 
liarities are  characteristic  when  grown  on  this  medium 
(see  p.  379).  The  bacilli  are  non-motile  and  Gram- 
positive.  The  colonies  are  round,  elievated,  smooth, 
and  grayish. 

9.  Typhoid  and  Colon  Bacilli. — These  are  medium-sized, 
motile,  Gram-negative,  non-spore-bearing  bacilh.  Upon 
blood-serum  they  form  rounded,  grayish,  slightly  ele- 
vated, viscid  looking  colonies,  those  of  the  colon  bacillus 
being  somewhat  the  larger.  They  do  not  liquefy  gela- 
tin. They  represent  the  extremes  of  a  large  group 
with  many  intermediate  types.  They  are  distinguished 
as  follows: 

27 


4l8  BACTERIOLOGIC  METHODS 

Typhoid  Bacillus.  Colon  Bacillus. 

Actively  motile.  Much  less  active. 

Growth  on  potato  usually  invisible.  Growth  distinctly  visible  as  dirty 

gray  or  brownish  slimy  layer. 

No  gas  produced  in  glucose  media.  Produces  gas. 

Growth  in  litmus  milk  produces  no  Litmus  milk  pink  and  coagulated. 

change. 

Produces  no  indol   in   Dunham's  Produces  indol.        (For  test,  see 

peptone  medium.  p.  404.) 

Agglutinates  with  serum  from  ty-  Does  not  agglutinate  with  typhoid 

phoid-fever  patient.      (Recently  serum. 

isolated  bacilli  do  not  agglutinate 

well.) 

10.  Bacillus  of  Influenza. — Diagnosis  will  usually  rest 
upon  the  morphology  and  staining  peculiarities,  described 
on  p.  58,  and  upon  the  fact  that  the  bacillus  will  not  grow 
on  ordinary  media,  but  does  grow  upon  hemoglobin-con- 
taining media.  It  can  be  grown  upon  agar-slants  which 
have  been  smeared  with  a  drop  of  blood  from  a  puncture 
in  the  finger.  Before  inoculation  these  slants  should  be 
incubated  to  make  sure  of  sterility.  The  colonies  are 
difficult  to  see  without  a  hand  lens.  They  are  very 
minute,  discrete,  and  transparent,  resembling  small 
drops  of  dew. 

11.  Bacillus  of  Tuberculosis. — The  methods  of  iden- 
tifying this  important  organism  have  been  given  (pp. 
49  and  168).  Cultivation  is  not  resorted  to  in  clinical 
work.  It  grows  very  slowly  and  only  on  certain  media. 
It  is  Gram-positive  and  non-motile. 


CHAPTER  DC 

PREPARATION  AND  USE  OF  VACCINES 

Bacterial  vaccines,  sometimes  called  "  bacterins," 
which  within  the  past  few  years  have  come  to  play  an 
important  role  in  therapeutics,  are  suspensions  of  defi- 
nite numbers  of  dead  bacteria  in  normal  salt  solution. 
While  in  many  cases,  notably  in  gonorrhea  and  tuber- 
culosis, ready  prepared  or  "  stock "  vaccines  are  satis- 
factory, it  is  usually  desirable  and  often  imperative  for 
best  results  to  use  vaccines  which  are  especially  prepared 
for  each  patient  from  bacteria  which  have  been  freshly 
isolated  from  his  own  lesion.  These  latter  are  called 
"  autogenous  vaccines."  Only  through  them  can  one 
be  certain  of  getting  the  exact  strain  of  bacterium  which 
is  producing  the  disease. 

I.  PREPARATION  OF  VACCINE 
The  method  of  preparing  autogenous  vaccines  which 
is  used  in  the  author's  laboratory  is  here  described. 

I.  Preparation  of  Materials. — A  number  of  2-ounce 
wide-mouthed  bottles  are  cleaned  and  sterilized.  Each  is 
charged  with  50  c.c.  freshly  filtered  normal  salt  solution 
(0.85  per  cent,  sodium  chlorid  in  distilled  water),  and  is 
capped  with  a  new  rubber  nursing-nipple,  without  holes, 
inverted  as  shown  in  Fig.  162.  A  small  section  of  hollow 
wire  or  a  hypodermic  needle  is  thrust  through  the  cap 
near  the  edge  to  serve  as  an  air  vent,  and  the  bottle  and 

419 


420 


PREPARATION   AND   USE   OP  VACCINES 


contents  are  sterilized  in  an  autoclave.  If  an  autoclave 
is  not  at  hand,  successive  steamings  in  an  Arnold  steril- 
izer will  answer,  provided  it  is  not  opened  between  steam- 
ings. After  sterilization,  the  pieces  of  wire  are  pulled 
out  and  the  holes  scaled  with  collodion. 


^CTERIALVA^ 


Fig.    162. — \'accine   bottles:    A,  Cap  ready   to   be   applied;    B,   ready  for  sterilization; 
C,  finished  vaccine. 


A  number  of  test-tubes,  each  charged  with  10  c.c.  of 
normal  salt  solution  and  plugged  with  cotton,  are  also 
prepared  and  sterilized. 

2.  Obtaining  the  Bacteria.— A  culture  on  some  solid 
medium  is  made  from  the  patient's  lesion,  and  a  pure 
culture  is  obtained  in  the  usual  way.  This  preliminary 
work  should  be  carried  through  as  quickly  as  possible. 
If  for  any  reason  there  is  much  delay,  it  is  best  to  begin 
over  again,  the  experience  gained  in  the  first  trial  en- 


PREPARATION   OF  VACCINE 


421 


abling  one  to  carry  the  second  through  more  rapidly. 
When  a  pure  culture  is  obtained,  a  number  of  tubes  of 
blood-serum  or  agar — 10  or  1 2  in  the  case  of  streptococ- 
cus or  pneumococcus,  4  or  5  in  the  case  of  most  other 
organisms — are  planted  and  incubated  over  night  or 
until  a  good  growth  is  obtained. 

3.  Making  the  Suspension. — The  salt  solution  from 
one  of  the  10  c.c.  salt-tubes  is  transferred  by  means  of  a 


Fig.  163. — Process  of  making  hermetically  sealed  capsules. 

sterile  pipet  to  the  culture-tubes,  and  the  growth  thor- 
oughly rubbed  up  with  a  stiff  platinum  wire  or  a  glass 
rod  whose  tip  is  bent  at  right  angles,  The  suspension 
from  the  different  tubes,  usually  amounting  to  about  10 
c.c,  is  then  collected  in  one  large  tube  (size  about  6  by 
I  inch) ;  and  the  upper  part  of  the  tube  is  drawn  out  in 
the  flame  of  a  blast  lamp  or  Bunsen  burner,  as  indicated 
in  Fig.  163,  a  short  section  of  glass  tubing  being  fused  to 


422 


PREPARATION   AND   USE   OF  VACCINES 


the  rim  of  the  tube  to  serve  as  a 
handle.  It  is  then  stood  aside,  and 
when  cool  the  end  is  sealed  off. 

The  resulting  hermetically  sealed 
capsule  is  next  thoroughly  shaken  for 
ten  to  twenty  minutes  to  break  up  all 
clumps  of  bacteria.  Some  small  pieces 
of  glass  or  a  little  clean  sterile  sand 
may  be  introduced  to  assist  in  this, 
but  with  many  organisms  it  is  not 
necessary. 

4.  Sterilization. — The  capsule  is 
placed  in  a  water-bath  at  60°  C.  for 
forty-five  minutes.  This  can  be  done 
in  an  ordinary  rice-cooker,  with  double 
lid  through  which  a  thermometer  is 
inserted.  When  both  compartments 
are  filled  with  water  it  is  an  easy 
matter  to  maintain  a  uniform  tem- 
perature by  occasional  application  of 
a  small  flame.  The  time  and  tem- 
perature are  important :  too  little  heat 
will  fail  to  kill  the  bacteria,  and  too 
much  will  destroy  the  efficiency  of 
the  vaccine. 

When  sterilization  is  complete  the 
capsule  is  opened,  a  few  drops  are 
planted  on  agar  or  blood-serum,  and 
the  capsule  is  again  sealed. 

5.  Counting. — When  incubation  of 
the  plant  has  shown  the  suspension  to 
be  sterile  it  is  ready  for  counting. 


PREPARATION   OF  VACCINE  423 

There  must  be  ready  a  number  of  clean  slides;  a  few 
drops  of  normal  salt  solution  on  a  slide  or  in  a  watch- 
glass;  a  blood-lancet, '  which  can  be  improvised  from  a 
spicule  of  glass  or  a  pen;  and  two  slender  pipets  with 
squarely  broken  off  tips  and  grease-pencil  marks  about 
2  cm.  from  the  tip  (Fig.  164).  These  are  easily  made  by 
drawing  out  a  piece  of  glass  tubing,  as  described  on  page 

398-  _ 

It  is  necessary  to  work  quickly.  After  thorough  shak- 
ing, the  capsule  is  opened  and  a  few  drops  forced  out 
upon  a  slide.  Any  remaining  clumps  of  bacteria  are 
broken  up  with  one  of  the  pipets  by  holding  it  against 
and  at  right  angles  to  the  slide,  and  alternately  sucking 
the  fluid  in  and  forcing  it  out.  The  pipet  is  most  easily 
controlled  if  held  in  the  whole  hand  with  the  rubber  bulb 
between  the  thumb  and  the  side  of  the  index-finger. 
A  finger  is  then  pricked  until  a  drop  of  blood  appears; 
and  into  the  second  pipet  are  quickly  drawn  successively : 
I  or  2  volumes  normal  salt  solution  (or  better,  a  i  percent, 
solution  of  sodium  citrate  which  prevents  coagulation); 
a  small  bubble  of  air;  i  volume  of  blood;  a  small  bubble  of 
air;  and,  finally,  i  volume  of  bacterial  suspension.  (A 
''volume"  is  measured  by  the  distance  from  the  tip  of 
the  pipet  to  the  grease-pencil  mark.)  The  contents  of 
the  pipet  are  then  forced  out  upon  a  slide  and  thoroughly 
mixed  by  sucking  in  and  out,  care  being  taken  to  avoid 
bubbles;  after  which  the  fluid  is  distributed  to  a  number 
of  slides  and  spread  as  in  making  blood-smears. 

The  films  are  stained  with  Wright's  blood-stain  or, 
better,  by  a  few  minutes'  application  of  carbol-thionin, 
after  fixing  for  a  minute  in  saturated  mercuric  chlorid 
solution.     With  an  oil-immersion  lens  both  the  red  cells 


424  PREPARATION    AND   USE   OF  VACCINES 

and  the  bacteria  in  a  number  of  microscopic  fields  are 
counted.  The  exact  number  .is  not  important;  for 
convenience  500  red  cells  may  be  counted.  P'rom  the  ra- 
tio between  the  number  of  bacteria  and  of  red  cells,  it 
is  easy  to  calculate  the  number  of  bacteria  in  i  c.c.  of 
the  suspension,  it  being  known  that  there  are  5000  million 
red  corpuscles  in  a  cubic  centimeter  of  normal  human 
blood.  If  there  were  twice  as  many  bacteria  as  red  cor- 
puscles in  the  fields  counted,  the  suspension  would  con- 
tain 10,000  million  bacteria,  per  cubic  centimeter. 

The  count  can  also  be  made  with  the  hemocytometer, 
using  a  weak  carbol-fuchsin  or  gentian  violet,  freshly 
filtered,  as  diluting  fluid.  A  very  thin  cover-glass  must 
be  used;  and,  after  filling,  the  counting-chamber  must 
be  set  aside  for  an  hour  or  more  to  allow  the  bacteria  to 
settle.  Mallory  and  Wright  advise  the  use  of  the  shallow 
chamber  manufactured  by  Zeiss  for  counting  blood- 
plates,  but  many  2  mm.  oil-immersion  objectives  have 
sufficient  working  distance  to  allow  the  use  of  the 
regular  Thoma  counting-chamber,  provided  a  very  thin 
cover  is  used. 

6.  Diluting. — The  amount  of  the  suspension,  which, 
when  diluted  to  50  c.c,  will  give  the  strength  desired  for 
the  finished  vaccine  having  been  determined,  this  amount 
of  salt  solution  is  withdrawn  with  a  hypodermic  syringe 
from  one  of  the  bottles  already  prepared,  and  is  replaced 
with  an  equal  amount  of  suspension.  One-tenth  c.c.  of 
trikresol  or  lysol  is  finally  added  and  the  vaccine  is 
ready  for  use.  To  prevent  possible  leakage  about  the 
cap,  the  neck  of  the  bottle  is  dipped  in  melted  paraffin. 
The  usual  strengths  are :  staphylococcus.  1000  million  in 
I  c.c;  most  other  bacteria,  100  million  in  i  c.c. 


DOSAGE  425 

II.  METHOD  OF  USE 

Vaccines  are  administered  subcutaneously,  usually  in 
the  arm  or  abdominal  wall  or  between  the  shoulder-blades. 
The  rubber  cap  is  sterilized  by  filling  the  concavity  with 
alcohol  for  some  minutes,  usually  while  the  syringe  is 
being  sterilized.  The  bottle  is  then  inverted  and  well 
shaken,  when  the  needle  is  plunged  through  the  rubber 
and  the  desired  quantity  withdrawn.  The  hole  seals 
itself  and  no  collodion  is  necessary,  which  is  one  of  the 
advantages  of  this  form  of  cap.  The  most  satisfactory 
syringe  is  the  comparatively  inexpensive  "  Sub-Q  Tuber- 
culin" syringe  graduated  in  hundredths  of  a  cubic  centi- 
meter. 

A  rapid  and  efficient  technic  for  giving  the  injections 
is  suggested  by  Major  F.  T.  Woodbury  of  the  Army 
Medical  Corps.  The  needle  is  dipped  into  tincture  of 
iodin  and  some  is  drawn  into  the  syringe  and  expelled; 
a  small  quantity  of  the  vaccine  or  of  sterile  water  is  like- 
wise drawn  in  and  expelled,  after  which  the  dose  to  be 
given  is  drawn  in.  The  patient's  arm  is  touched  with  a 
swab  of  cotton  saturated  with  tincture  of  iodin  and  the 
injection  is  made  through  the  resulting  brown  spot. 
The  syringe  is  cleaned  by  drawing  into  it  and  expeUing, 
successively,  tincture  of  iodin,  alcohol,  and  air. 

III.  DOSAGE 

-Owing  to  variations,  both  in  virulence  of  organisms  and 
susceptibility  of  patients,  no  definite  dosage  can  be 
assigned.  Each  case  is  a  separate  problem.  Wright's 
original  proposal  was  to  regulate  the  size  and  frequency 
of  dose  by  its  effect  upon  the  opsonic  index,  but  this  is 


426  PREPARATION   AND  USE   OF  VACCINES 

beyond  the  reach  of  the  practitioner.  The  more  widely 
used  "clinical  method"  consists  in  beginning  with  a 
very  small  dose  and  cautiously  increasing  until  the 
patient  shows  either  improvement  or  some  sign  of  a 
"reaction,"  indicated  by  headache,  malaise,  fever,  ex- 
acerbation of  local  disease,  or  inflammatory  reaction  at 
the  site  of  injection.  The  reaction  indicates  that  the 
dose  has  been  too  large.  The  beginning  dose  of  staphy- 
lococcus is  about  50  million;  the  maximum,  1000  million 
or  more.  Of  most  other  organisms  the  beginning  dose 
is  5  million  to  10  million;  maximum,  about  100  million, 
Ordinarily,  injections  are  given  once  or  twice  a  week; 
very  small  doses  may  be  given  every  other  day. 

IV.  THERAPEUTIC  INDICATIONS 

The  therapeutic  effect  of  vaccines  depends  upon  their 
power  to  stimulate  the  production  of  opsonins  and  other 
antibacterial  substances  which  enable  the  body  to  com- 
bat the  infecting  bacteria.  Their  especial  field  is  the 
treatment  of  subacute  and  chronic  localized  infections, 
in  some  of  which  they  offer  the  most  effective  means  of 
treatment  at  our  command.  In  most  chronic  infections 
the  circulation  of  blood  and  lymph  through  the  diseased 
area  is  very  sluggish,  so  that  the  antibodies,  when  formed, 
cannot  readily  reach  the  seat  of  disease.  Ordinary 
measures  which  favor  circulation  in  the  diseased  part 
should,  therefore,  accompany  the  vaccine  treatment. 
Among  these  may  be  mentioned  incisions  and  drainage 
of  abscesses,  dry  cupping,  application  of  heat.  Bier's 
hyperemia,  etc.,  but  such  measures  should  not  be  applied 
during  the  twenty-four  hours  succeeding  an  injection, 
since  the  first  effect  of  the  vaccine  may  be  a  temporary 


THERAPEUTIC  INDICATIONS  427 

lowering  of  resistance.  Vaccines  are  of  little  value,  and, 
in  general,  are  contraindicated  in  very  acute  infections, 
particularly  in  those  which  are  accompanied  by  much 
systemic  intoxication,  for  in  such  cases  the  power  of  the 
tissues  to  produce  antibodies  is  already  taxed  to  the  limit. 
It  is  true,  nevertheless,  that  remarkably  beneficial  results 
have  occasionally  followed  their  use  in  such  acute  condi- 
tions as  malignant  endocarditis,  but  here  they  should 
be  tried  with  extreme  caution. 

Probably  best  results  are  obtained  in  staphylococcus 
infections,  although  pneumococcus,  streptococcus,  and 
colon  bacillus  infections  usually  respond  nicely.  Among 
clinical  conditions  which  have  been  treated  successfully 
with  vaccines  are  furunculosis,  acne  vulgaris,  infected 
operation- wounds,  pyelitis,  cystitis,  subacute  otitis 
media,  osteomyelitis,  infections  of  nasal  accessory  si- 
nuses, etc.  Vaccine  treatment  of  the  mixed  infection  is 
doubtless  an  important  aid  in  tuberculosis  therapy, 
and  in  some  cases  the  result  is  brilliant.  When,  as  is 
common,  several  organisms  are  present  in  the  sputum, 
a  vaccine  is  made  from  each,  excepting  the  tubercle 
bacillus,  'of  which  autogeneous  vaccines  are  not  used  in 
practice.  To  avoid  the  delay  and  consequent  loss  of 
virulence  entailed  by  study  and  isolation  of  the  several 
varieties,  many  workers  make  the  bacterial  suspension 
directly  from  the  primary  cultures.  The  resulting  vac- 
cines contain  all  strains  which  are  present  in  the  sputum 
in  approximately  the  same  relative  numbers.  Although 
open  to  criticism  from  a  scientific  standpoint,  this 
method  offers  decided  practical  advantages  in  many 
cases. 

It  has  been  shown  that  vaccines  are  useful  in  prevent- 


428  PREPARATION   AND   USE   OF   VACCINES 

ing  as  well  as  curing  infections.  Their  value  has  been 
especially  demonstrated  in  typhoid  fever.  Three  or  four 
doses  of  about  50,000,000  typhoid  bacilli  are  given  about 
seven  days  apart.  Results  in  the  army,  where  the  plan 
has  been  tried  on  a  large  scale,  show  that  such  vaccina- 
tion is  effective. 

V.  TUBERCULINS 

Tuberculins  contain  the  products  of  tubercle  bacilli 
or  their  ground-up  bodies,  the  latter  class  being  prac- 
tically vaccines.  They  are  undoubtedly  of  great  value 
in  the  treatment  of  localized  tuberculosis,  particularly 
of  bones,  joints,  and  glands;  and  are  of  rather  indefmite 
though  certainly  real  value  in  chronic  pulmonary  tuber- 
culosis, especially  when  quiescent.  The  best  known  are 
Koch's  old  tuberculin  (T.  O.),  bouillon  filtrate  (B.  F.), 
triturate  residue  (T.  R.),  and  bacillary  emulsion  (B.  E.). 
There  seems  to  be  little  difference  in  the  actions  of  these, 
although  theoretically  T.  R.  should  immunize  against 
the  bacillus  and  B.  F.  against  its  toxic  products.  The 
choice  of  tuberculin  is  much  less  important  than  the 
method  of  administration.  The  making  of  autogenous 
tuberculins  is  impracticable,  hence  stock  preparations 
are  used  in  practice. 

Since  the  dose  is  exceedingly  minute,  the  tuberculin 
as  purchased  must  be  greatly  diluted  before  it  is  avail- 
able for  use.  A  convenient  plan  is  to  use  the  rubber- 
capped  50  c.c.  bottles  of  sterile  salt  solution  described 
for  vaccines  (p.  419),  adding  sufficient  tuberculin  to  give 
the  desired  strength,  together  with  o.i  c.c.  trikresol  to 
insure  sterility.  The  practitioner  should  bear  in  mind 
that  while  tuberculin  is  capable  of  good,  it  is  also  capable 


TUBERCULIN  IN  DIAGNOSIS  429 

of  great  harm.  Everything  depends  upon  the  dosage 
and  plan  of  treatment.  Probably  a  safe  beginning  dose 
for  a  pulmonary  case  is  0.0000 1  milligram  of  B.  E.,  B.  F., 
or  T.  R. ;  for  gland  and  bone  cases,  about  o.oooi  milligram. 
O.  T.  is  now  used  chiefly  in  diagnosis.  The  intervals  are 
about  one  week  or,  rarely,  three  days,  when  very  small 
doses  are  given.  The  dose  is  increased  steadily,  but  with 
extreme  caution;  and  should  be  diminished  or  temporarily 
omitted  at  the  first  indication  of  a  "reaction,"  of  which, 
in  general,  there  are  three  forms : 

(a)  Getter al:  Elevation  of  temperature  (often  slight), 
headache,  malaise. 

(6)  Local:  Increase  of  local  symptoms,  amount  of 
sputum,  etc. 

(c)  Stick:  Inflammatory  reaction  at  site  of  injection. 

Treatment  is  usually  continued  until  a  maximum  dose 

of  I  mm.  is  reached,  the  course  extending  over  a  year  or 

more. 

VI.  TUBERCULIN  IN  DIAGNOSIS 

The  tissues  of  a  tuberculous  person  are  sensitized 
toward  tuberculin,  and  a  reaction  (see  preceding  section) 
occurs  when  any  but  the  most  minute  quantity  of  tuber- 
cuUn  is  introduced  into  the  body.  Non-tuberculous 
persons  exhibit  no  such  reaction.  This  is  utilized  in 
the  diagnosis  of  obscure  forms  of  tuberculosis,  the 
test  being  applied  in  a  number  of  ways: 

I.  Hypodermic  Injection. — Koch's  old  tuberculin  is 
used  in  successive  doses,  several  days  apart,  of  o.ooi ,  o.oi, 
and  0.1  mg.  A  negative  result  with  the  largest  amount 
is  considered  final.  The  reaction  is  manifested  by 
fever  within  eight  to  twenty  hours  after  the  injection. 
The  method    involves    some    danger  of   lighting  up  a 


430  PREPARATION  AND  USE   OF   VACCINES 

latent  process,  and  has  been  largely  displaced  by  safer 
methods. 

2.  Calmette's  Ophthalmo-reaction. — One  or  two  drops 
of  0.5  per  cent,  purified  old  tuberculin  are  instilled  into 
one  eye.  Tuberculin  ready  prepared  for  this  purpose  is 
on  the  market.  If  tuberculosis  exists  anywhere  in  the 
body,  a  conjunctivitis  is  induced  within  twelve  to  twenty- 
four  hours.  This  generally  subsides  within  a  few  days. 
The  method  is  not  without  some,  though  sUght,  risk  of 
injury  to  the  eye;  and  the  test  is  absolutely  contraindi- 
cated  in  the  presence  of  any  form  of  ocular  disease, 
A  second  instillation  should  not  be  tried  in  the  same 
eye. 

3.  More  Reaction. — A  50  per  cent,  ointment  of  old  tu- 
berculin in  lanolin  is  rubbed  into  the  skin  of  the  abdomen, 
a  piece  about  the  size  of  a  pea  being  required.  Dermati- 
tis, which  appears  in  twenty-four  to  forty-eight  hours, 
indicates  a  positive  reaction.  The  ointment  can  be 
purchased  ready  for  use. 

4.  Von  Pirquet's  Method. — This  is  the  most  satis- 
factory of  the  tuberculin  tests.  Two  small  drops  of 
old  tuberculin  are  placed  on  the  skin  of  the  front  of  the 
forearm,  about  2  inches  apart,  and  the  skin  is  slightly 
scarified,  first  at  a  point  midway  between  them,  and  then 
through  each  of  the  drops.  A  convenient  scarifier  is  a 
piece  of  heavy  platinum  wire,  the  end  of  which  is  ham- 
mered to  a  chisel  edge.  This  is  held  at  right  angles  to 
the  skin,  and  rotated  six  to  twelve  times  with  just  suffi- 
cient pressure  to  remove  the  epidermis  without  drawing 
blood.  In  about  ten  minutes  the  excess  of  tuberculin 
is  gently  wiped  away  with  cotton.  No  bandage  is  neces- 
sary.    A  positive  reaction  is  shown  by  the  appearance  in 


TUBERCULIN  IN  DIAGNOSIS  43 1 

twenty-four  to  forty-eight  hours  of  a  papule  with  red 
areola,  which  contrasts  markedly  with  the  small  red  spot 
left  by  the  control  scarification. 

These  tests  have  very  great  diagnostic  value  in  chil- 
dren, especially  those  under  three  years  of  age,  but  are 
often  misleading  in  adults,  positive  reactions  occurring 
in  many  apparently  healthy  individuals.  Negative  tests 
are  very  helpful  in  deciding  against  the  existence  of 
tuberculosis. 


APPENDIX 

L  APPARATUS,  REAGENTS,  AND  STAINS 

The  apparatus  and  reagents  listed  here  are  sufficient 
for  all  but  the  rarer  tests  described  in  the  text.  Those  in 
smaller  type  are  less  frequently  required.  For  ordinary 
routine  work  a  much  smaller  list  will  suffice. 

A,  APPARATUS 

Beakers  and  flasks,  several  sizes,  preferably  of  Jena 
glass. 

Blood  lancet,  or  some  substitute  (Fig.  68). 

Bunsen-burner  or  alcohol  lamp. 

Buret,  25  c.c.  capacity,  preferably  with  Schellbach 
stripe. 

Buret  and  filter-stand  combined. 

Centrifuge — hand,  electric,  or  water-power  (Figs.  20 
and  21).  With  the  last  two  a  speed  indicator  is  desirable. 
Radius  of  arm  when  in  motion  should  be  6f  inches. 
Plain  and  graduated  tubes  accompany  the  instrument ; 
milk- tubes  (Fig.  157)  must  be  purchased  separately.  The 
hematocrit  attachment  (Fig.  77)  is  not  much  used. 

Cigaret-paper,  "Zig-zag"  brand  or  some  similar  thin 
paper,  for  making  blood-films. 

Corks,  preferably  of  rubber,  with  one  and  two  holes. 

Cover-glasses,  No.  2  thickness — |-inch  squares  are 
most  convenient. 

Cover-glass  forceps. 

432 


APPARATUS  433 

Esbach's  tube  (Fig.  27). 

Evaporating  dish. 

Filter-paper:  ordinary  cheap  paper  for  urine  filtration; 
"ashless"  quantitative  filter-paper  for  chemic  analyses. 

Glass  funnels. 

Glass  rods  and  tubing  of  sodium  glass :  for  stirring  rods, 
urinary  pipets,  etc. 

Glass  slides:  the  standard  i-  by  3-inch  size  will  answer 
for  all  work,  although  a  few  larger  slides  will  be  found 
convenient;  those  of  medium  thickness  are  preferable. 

Graduates,  cylindric  form,  several  sizes. 

Graniteware  basin. 

Hemoglobinometer :  see  pp.  185  to  191  for  descriptions 
of  the  different  instruments. 

Hemocy tometer :  either  Tiirck  or  Zappert  ruling  is 
desirable  (Figs.  73,  74,  and  79). 

Hypodermic  syringe:  the  "Aseptic  Sub-Q,  Tubercu- 
lin," is  probably  the  most  useful  type. 

Incubator  (p.  397). 

Labels  for  slides  and  bottles. 

Litmus-paper,  red  and  blue,  Squibb's  preferred. 

Mett's  tubes  (pp.  300  and  399). 

Microscope  (Fig.  i).     Equipment  described  on  p.  31. 

Petri  dishes. 

Platinum  wires  (p.  397). 

Sterilizers:  the  Arnold  type  for  steaming;  oven  for  dry 
sterilization  (p.  396). 

Stomach-tube. 

Test-glass,  conic  jOne  side  painted  half  white,  half  black . 

Test-tubes,  rack,  and  cleaning  brush. 

Ureometer,  Doremus-Hinds'  pattern  (Fig.  24). 

Urinometer,  preferably  Squibb's  (Fig.  17). 

28 


434  APPENDIX 

Blood-fixing  oven,  or  Kowarsky's  plate  (Fig.  84). 

Copper-foil  and  gauze. 

Cotton,  absorbent,  for  filtering,  etc. 

"  Cotton-batting  "  for  plugging  tubes. 

Culture-media.  The  selection  depends  upon  the  work 
to  be  done  (p.  401). 

Holt's  cream  gage  and  hydrometer  (Fig.  156). 

Horismascope  (Fig.  26). 

Pipets,  graduated,  5  to  50  c.c.  capacity. 

Ruhemann's  tube  for  uric-acid  estimation  (Fig.  25). 

Saccharimeter  (Fig.  29). 

Strauss'  separatory  funnel  for  lactic-acid  test  (Fig.  105). 

Suction  filter. 

Urinopyknometer  of  Sa.xe  (Fig.  18). 

Widal  reaction  outfit:  either  living  agar  cultures  of  the 
t>-phoid  bacillus,  or  the  dead  cultures  with  diluting  apparatus, 
which  are  sold  under  various  trade  names. 

Water-bath. 

B.  REAGENTS  AND  STAINS 

All  stains  and  many  reagents  are  best  kept  in  small 
dropping  bottles.  Formulae  are  given  in  the  text.  Dry 
stains  (Griibler's  should  be  specified)  and  most  staining 
solutions  and  chemical  reagents  can  be  purchased  of 
Bausch  &  Lomb  Optical  Co.,  Rochester,  New  York,  or 
Eimer  &  Amend,  New  York.  For  the  physician  who 
does  only  a  small  amount  of  work,  the  "  Soloid  "  tablets 
manufactured  by  Burroughs.  Wellcome  &  Co.  are  con- 
venient and  satisfactory.  These  tablets  have  only  to 
be  dissolved  in  a  specified  amount  of  fluid  to  produce 
the  finished  stain.  IMost  of  the  stains  mentioned  here 
come  in  this  form. 

Acid,  glacial  acetic.  Other  strengths  can  be  made 
from  this  as  desired. 


REAGENTS  AND  STAINS  435 

Acid,  hydrochloric,  concentrated  (contains  about  32 
per  cent,  by  weight  of  absolute  hydrochloric  acid). 
Other  strengths  can  be  made  as  desired. 

Acid,  nitric,  strong,  colorless. 

Acid,  nitric,  yellow.  Can  be  made  from  colorless  acid 
by  adding  a  splinter  of  pine,  or  allowing  to  stand  in 
sunlight. 

Acid,  sulphuric,  concentrated. 

Alcohol,  ethyl  (grain-alcohol) .  This  is  ordinarily  about 
93  to  95  per  cent.,  and  other  strengths  can  be  made  as 
desired. 

Aqua  ammoniae  fortior  (sp.  gr.  0.9). 

Bromin,  or  Rice's  solutions  (p.  95),  for  urea  estimation. 

Chloroform. 

Diluting  fluid  for  erythrocyte  count  (p.  198). 

Diluting  fluid  for  leukocyte  count  (p.  213). 

Dimethyl-amido-azobenzol,  0.5  per  cent,  alcoholic 
solution. 

Distilled  water. 

Esbach's  or  Tsuchiya's  reagent  (p.  105). 

Ether,  sulphuric. 

Ferric  chlorid:  saturated  aqueous  solution  and  10  per 
cent,  aqueous  solution. 

Haines'  (or  FehHng's  or  Benedict's)  solution  (pp.  109, 
no). 

Lugol's  solution  {Liquor  lodi  Compositus,  U.  S.  P.). 
Gram's  iodin  solution  (p.  57)  can  be  made  from  this  by 
adding  fourteen  times  its  volume  of  water. 

Obermayer's  reagent  (p.  91). 

Phenylhydrazin,  pure. 

Phenol. 

Phenolphthalein,  i  or  0.5  per  cent,  alcoholic  solution. 


436  APPENDIX 

Purdy's  (or  Fehling's  or  Benedict's)  solution  (pp.  112- 

115). 

Robert's  reagent  (p.  103). 

Sodium  chlorid  (table  salt),  saturated  aqueous  solu- 
tion. 

Sodium  hydroxid  (caustic  soda) ,  40  per  cent,  solution ; 
other  strengths  can  be  made  from  this  as  desired. 

Sodium  hydroxid,  decinormal  solution.  The  prac- 
titioner will  find  it  best  to  purchase  this  solution  ready 
prepared.  Eimer  and  Amend,  New  York,  and  many 
other  chemical  supply  houses  carry  it  in  stock.  For 
ordinary  cUnical  work  41  grams  of  Merck's  "  sodium 
hydrate  by  alcohol "  from  a  freshly  opened  bottle  may 
be  dissolved  in  icxx)  c.c.  water.  This  makes  a  normal 
solution  and  must  be  diluted  with  9  volumes  of  water  to 
make  the  decinormal  solution. 

Sodium  nitrite,  0.5  per  cent,  solution  for  diazo-reaction. 
Must  be  freshly  prepared. 

Sulphanilic  acid  solution  for  diazo-reaction  (p.  128). 

Stains: 

Carbol  fuchsin  (p.  50). 

Eosin,  saturated  aqueous  solution. 

Formalin  -  gentian  -  violet,   or  anihn  -  gentian  -  violet 

(P-  57)- 
Gabbet's    stain    or    Pappenheim's    methylene-blue 

stain  (p.  51). 
Loffler's  alkaline  methylene-blue  solution  (p.  57). 
Pappenheim's  pyronin-methyl-green  stain  (p.  408). 
Stain   for   fat:    Sudan   III.    saturated    solution   in 

70  per  cent,   alcohol;   or    i   per   cent,   aqueous 

solution  osmic  acid. 
Wright's  or  Harlow's  stain  for  blood. 


REAGENTS   AND   STAINS  437 

Tincture  of  guaiac,  diluted  to  a  light  sherry-wine  color 
(keep  in  a  dark  glass  bottle). 
Turpentine,  "ozonized"  (p.  125). 

Acid,  boric,  for  preserving  urine  (p.  69). 

Acid,  oxalic. 

Acid,  salicylous  (salicyl  aldehyd),  10  per  cent,  alcoholic 
solution. 

Alcohol,  amylic. 

Alcohol,  ethyl,  absolute. 

Alcohol,  methyl  (pure). 

Antiformin  (p.  52). 

Barium  chlorid  mixture  (p.  89). 

Benzol. 

Boas'  reagent  or  Gunzburg's  (p.  290). 

Boggs'  reagent  (p.  387). 

Calcium  chlorid,  i  per  cent,  solution. 

Canada-balsam  in  xylol:  necessary  only  when  permanent 
microscopic  preparations  are  made. 

Carbon  disulphid. 

Charcoal,  animal. 

Chromium  trioxid. 

Congo-red,  strong  alcoholic  solution. 

Copper  sulphate. 

Diluting  fluid  for  blood-platelet  count  (pp.  215,  216). 

Egg-albumen  discs  in  glycerin  (p.  293). 

Ether,  acetic,  pure, 

Florence's  reagent  (p.  393). 

Formalin  (40  per  cent,  solution  of  formaldehyd  gas). 
•     India-ink  (Gunther  and  Wagner)  (p.  391). 

lodin  crystals. 

Iron  sulphid. 

Lead  acetate  (sugar  of  lead) ;  used  in  10  per  cent,  solution 
to  clarify  urine. 


438  APPENDIX 

Lead  acetate,  tribasic. 
Lime-water. 

Miiller's  fluid  saturated  with  mercuric  chlorid  (p.  56). 
Orcin. 

Pepsin,  U.  S.  P. 
Phenylhydrazin  hydrochlorid. 
Potassium  ferrocyanid,  10  per  cent,  solution. 
Potassium  oxalate  (neutral). 
Potassium  persulphate. 
Ruhemann's  reagent  (p.  97). 

Silver  nitrate  crystals;  also  dram  to  the  ounce  aqueous 
solution,  and  "  ammoniated  "  solution  (p.  97). 

Sodium  alizarin  sulphonate,  i  per  cent,  aqueous  solution. 
Sodium  carbonate. 

Sodium  chlorid,  2  per  cent,  solution ;  from  this  normal  salt 
solution  (0.8  per  cent.)  can  be  made  as  desired. 
Sodium  hyposulphite. 
Sodium  nitroprussid. 
Sodium  sulphate. 
Stains: 

Bismarck-brown,  saturated  aqueous  or  alcoholic  solution. 

Carbol-thionin. 

Ehrlich's  triple  stain  for  blood. 

Eosin,  0.5  per  cent,  alcoholic  solution  for  blood. 

Fuchsin,  weak  solution;  can  be  made  when  desired  by 
adding  a  little  carbol-fuchsin  to  a  test-tube  of  water. 

Gentian- violet,  saturated  alcoholic  solution. 

Giemsa's  stain  (p.  390). 

Methylene-blue  and  borax  solution  (p.  254). 

Methylene-blue,  saturated  aqueous  solution  for  blood. 

Van  Gieson's  stain  for  Negri  bodies  (p.  395). 
Sulphur,  powdered. 

Talc,  purified  (Talcum  Purificatum,  U.  S.  P.). 
Trichloracetic  acid  solution  (p.  102). 


WEIGHTS   AND  MEASURES 


439 


Uranium  nitrate,  5  per  cent,  aqueous  solution. 

Xylol. 

Zinc,  arsenic  free. 


IL  WEIGHTS,    MEASURES,  ETC,  WITH 
EQUIVALENTS 


Meter  (unit  of  length) : 

Gram  (unit  of  weiglit) : 
Liter  (unit  of  capacity) : 


METRIC 

Millimeter  (mm.)  =  j^bb  meter. 
Centimeter  (cm.)  =  yjg  meter. 
Kilometer  =  looo  meters. 


Micron  (/x) 


=  xiAiff  millimeter. 


Milligram  (mg.)    =  ^o^jj  gram. 
Kilogram  (kilo.)    =  looo  meters. 

Cubic  Centimeter  =  jo'jj  liter.      Same   measure  as  milli- 
liter  (ml.). 


I  Millimeter  = 
I  Centimeter  ^ 
I  Meter  = 


0-03937  (5's  approx.)  in. 
1000  microns. 
0.3937  (S  approx.)  in. 
0.0328  feet. 

39-37  in- 
3.28  feet. 
!b4d 


X  Micron  (M)={^^7^"aU^,t„, 


I  Sq.  Millimeter  =  0.00155"! 

1  Sq.  Centimeter  =  0.1550    >sq.  in. 

1  Sq.  Meter  =  1550      j 

1  Sq.  Meter  =  10.76  sq.  feet. 


1  Inch  =  25.399  millimeters. 

I  Sq.  Inch  =  6.451  sq.  centimeters. 
1  Cu.  Inch  =  16.387  cu.  centimeters. 


I  Gram 


I  Kilogram  = 


X  Liter 


15-43  grams. 

0.563  dram     "j 

0.035  ounce     >  Avoir. 

0.0022  pound) 

0.257  dram      l 

0.032  ounce     >Apoth. 

0.0027  pound  1 

35.27  ounce  (Avoir.). 

2.2  pound  (Avoir.), 
f  1.056  (i  approx.)  quart. 
^■<  61.02  cu.  inches. 
(1000  cu.  centimeters. 


I  Cu.  Millimeter  =  0.00006 )         . 
1  Cu.  Centimeter  =  0.0610  J  " 

1  Cu.  Centimeter  =  o.ooi  liter. 

,.32  cu.  feet. 

1025.4  cu.  iu. 


I  Cu.  Meter 


135-3 
\6io: 


I  Foot         =  30.48  centimeters. 
I  Sq,  Foot  =  0.093  sq.  meter. 
I  Cu.  Foot  =  0.028  cu.  meter. 


AVOIRDUPOIS   WEIGHT 


'  Ounce  =  {437.5^^f- 
1  Pound  =  16  oimces. 


I  Grain    =  0.065  (^  approx.)     "I 

I  Dram   =  1.77  (ij  approx.)        1  _._s 

I  Ounce  =  28.35  (30  approx.)      f° 

I  Pound  =  453-59  (500  approx.)  J 

I  Pound  =  27.7  cu.  inches. 

1  Pound  =  1.2151b.  Troy. 


APOTHECARIES'   MEASURE 


I  Dram   =  60  minims. 
1  Ounce  =  8  drams. 
'I  Pint      =  16  ounces. 
I  Gallon  =  8  pints. 


I  Dram   =  3.70 

I  Ounce  =  29.57 

1  Pint      =  473.1 

X  Gallon  =  3785.4 

I  Gallon  =  231  cu.  inches 


cu.  centimeters. 


440 


APPENDIX   . 


APOTHECARIES'   WEIGHT 


I  Scrup 

e  = 

=  20  gra 

ns. 

I  Grair 

= 

-  0.065  1 

I  Dram     =  3  scruples  =  60 

grains 

I  Uram  =  3  887  1   „„„, 
I  Ounce  =3110  [^a""*- 
I  Pound  =^  373.2  J 

I.  Ounce   =  8  drams -=  480  grains. 

I  Pound  -—  12  ounces. 

To  convert         minims 

into  cubic  centimeters  multiply  by   0.061 
"    cubic  centimeters        "             29.57 

"          jluidounces 

"         "               grains 

" 

grams                   "         "     0.0648 

"         "               drams 

" 

grams                 "         "     3.887 

"         "      cubic  centimeters 

minims                 "          "    16.23 

"         "      cubic  centimeters 

jluidounces             "         "     0.0338 

"          "                 grams 

grains                  "         "   15.432 

"         "               grams 

drams                 "         "     0.257 

TEMPERATURE 

Centigrade.                    Fahrenheit. 

Centigrade.                Fahrenheit. 

110° 230° 

37° 98.6° 

100 

212 

36.5 

•    •    97-7 

95 

203 

36 

.    ,    96.8 

90 

194 

35-5    • 

•    •    959 

85 

185 

35 

•    •    95 

80 

176 

34 

•    93-2 

75 

167 

33 

•    •    914 

70 

•158 

32 

89.6 

65 

149 

31 

.    .    87.8 

60 

140 

3" 

.    .    86 

55 

131 

25 

•    •     77 

50 

122 

20 

.    .    68 

45 

"3 

15 

•    ■    59 

44 

I  I  1.2 

10 

•    ■     50 

43 

109.4 

+5 

.    .    41 

42 

107.6 

0 

•    32 

41 

105.8 

—5 

.    .    .     23 

40.5 

104.9 

— 10 

.    .     14 

40 

104 

—  15 

•    ■  45 

39-5 

103. 1 
102.2 

— 20      — 4 

39 

38.5 

IOI.3 

0.54°        =-        1° 

■         38 

100.4 

I              =        1.8 

37-5 

99-5 

2              =        3.6 

25 

=        4-5 

To  convert  Fahrenheit  into  Centigrade,  subtract  32  and 
multiply  by  0.555. 

To  convert  Centigrade  into  Fahrenheit,  multiply  by 
1.8  and  add  32. 


INDEX 


Absorption,  toxic,  degree  of,  236 
Absorptive  power  of  stomach,  306 
Accidental  albuminuria,  100 
Acetanilid  in  urine,  132 
Acetic  acid  in  gastric  contents,  291 
Acetone  in  urine,  118.       See  also 

Acetomiria. 
Acetonuria,  118 

after  anesthesia,  119 

detection,  119 

Frommer's  test  in,  122 

Gunning's  test  in,  120 

Lange's  test  in,  121 

Legal's  test  for,  Lange's  modifi- 
cation, 121 

Lieben's  test  in.  Gunning's  modi- 
fication, 121 

tests,  1 19-122 
Achard  and  Castaigne's  methylene- 

blue  test  for  urine,  79 
Achlorhydria,  297 
Acholic  stools,  312 
Achromatic  objectives,  23 
Achylia  gastrica,  gastric  contents 

in,  304 
Acid  deficit  of  gastric  contents,  299 

intoxication,  cause,  118 
Acid-fast  bacilli,  53 
Acidity  of  urine,  quantitative  esti- 
mation, 74 
Folin's  method,  74 
Acidophilic  structures  of  blood,  221 
Actinomyces  bovis  in  sputum,  46 
Agar-agar,  glycerin,  preparation  of, 
402 

preparation  of,  402 
Agglutination,  257 
Agglutinins,  257 
Air-bubbles  in  urine,  173 
Albumin  in  sputum,  63 


Albumin  in  urine,  99.       See  also 

Albuminuria. 
AJbuminometer,  Esbach's,  105 
Albuminuria,  99,  100 

accidental,  100 

centrifugal   estimation   of   albu- 
min, 106 

cyclic,  100 

detection,  102 

Esbach's  estimation  of  albumin, 

estimation  of  albumin  in,  quan- 
titative, 105 

false,  100 

from  blood  changes,  loi 

from  kidney  changes,  loi 

heat  and  nitric  acid  test  in,  104 
test  in,  Purdy's,  104 

nitric  acid  test  in,  104 

orthostatic,  100 

physiologic,  100 

postural,  100 

Purdy's  centrifugal  method,  106 
heat  test  in,  104 
table  after. centrifugation,  107 

renal,  100 

Robert's  test  in,  103 

tests,  102-106 

trichloracetic  acid  test  in,  102 

Tsuchiya's   estimation   of   albu- 
min, 105 
Alkaline  methylene-blue,  LoflSer's, 

57 
phosphates  in  urine,  87 
urine,  unorganized  sediments  in, 
148 
Alkapton  bodies  in  urine,  1 26 
Alkaptonuria,  126 
Alveolar  cells  in  sputum,  62 
Amboceptor,  265,  266,  267 

441 


442 


INDEX 


Amboceptor,  hemolytic,  269 
Ameba?,  328.     See  also  Entamceba. 
Ameboid    movements   of   malarial 

parasites,  248 
Amidobenzol   test   for  free  hydro- 
chloric acid,  290 
Ammonia  in  urine,  97 
Brown's  test.  99 
decreased,  98 

estimation,  quantitative,  98 
increased,  98 

Ronchese-Malfatti       formalin 
test,  98 
Amnioniated  silver  nitrate  solution, 

97 
Ammoniomagnesium        phosphate 

crystals  in  urine,  148 
Ammonium  urate  crystals  in  urine, 

151 
Ammon's  horn,  394 
Amoeba  histolytica  in  sputum,  48 
Amorphous  phosphates  in  urine,  72, 

87,  149 
in  mass,  160 

urates  in  urine,  72,  143 
in  mass,  160 
Anaemia  infantum  pseudoleukaem- 

ica,  282 
Anemias,  275 

aplastic,  279 

blood-picture  in,  276 

blood-plaques  in,  214 

color  index  in,  200 

degeneration  of  Grawitz  in,  228 

erythroblasts  in  blood  in,  230 

erythrocytes  in,  226 

leukopenia  in,  202 

lymphocytes  in,  234 

myelocj'tes  in,  242 

oligocythemia  in,  193 

pernicious,  277 

p)olychromatophilia  in,  227 

primary',  277 

secondary-,  276 

splenic,  280 
Anesthesia,  acetonuria  after,  119 
Angina,  \'incent's,  379 
spirochaete  of,  331 
Anguillula,  354 

aceti,  354 
in  urine,  171 
Anilin  dyes  for  blood-films,  220 


Anilin-gentian- violet  stain,  57 

Animal  inoculation,  375 

method  for  tubercle  bacillus  in 

sputum,  53 
of  bacteria,  415 
parasites,  323.       See  also  Para- 
sites, animal. 

Anopheles,  248,  250 

Antibodies,  265,  269 

Antiformin    method    for    bacillus 
tuberculosis  in  sputum,  52 

Antigen,  269 

Antipyrin  in  urine,  132 

Anuria,  71 

Aplastic  anemia,  279 

Apochromatic  objectives,  23 

Apothecaries'  measure,  439 
weight,  440 

Apparatus,  396,  432 

Appendicitis,  leukocytosis  in,  206 

Arsenic  in  urine,  133 

Gutzeit's  test  for,  133 
Reinsch's  test  for,  133 
poisoning,  anemia  from,  276 

Arthropoda,  366 

Ascaris.  354 
lumbricoides,  354 
ova  of,  355 

Asparagus,  odor  of  urine  from,  73 

Asthma,  bronchial,  eosinophilia  in, 

239 
sputum  in,  66 
Atrophic  gastritis,  gastric  contents 

in,  305 
Atropin  in  urine,  133 
Autoclave,  396 
Autogenous  vaccines,  419 
Avoirdupois  weight,  439 


Babcock  estimation  for  fat  in  milk, 

386^ 
Babesia,  339 
bigeminum,  339 
hominis,  339 
Bacillus,  acid-fast,  53 

Boas-Oppler,  in  feces,  318 

in  gastric  contents,  303 
colon,  417 

in  otitis,  383 
diphtheria,  417 

in  eye  affections,  382 


INDEX 


443 


Bacillus,  diphtheria,  in  mouth,  378 
Koch-Weeks,    in    conjunctivitis, 

381 
mucosus  capsulatus  in  sputum, 

of  Friedlander  in  otitis,  383 

in  sputum,  58 
of  influenza,  418 

in  spinal  fluid,  376 

in  sputum,  58 
of  Vincent's  angina,  380 
pyocyaneus  in  otitis,  383 
smegma,  53,  169 
tuberculosis,  418 

in  cerebrospinal  fluid,  374 

in  feces,  319 

in  otitis,  383 

in  pus,  369 

in  sputum,  36,  49 
animal  inoculation  method, 

53 
antiformin  method,  52 
examination,  36 
Gabbet's  method,  49 
methods  for,  49-53 
Pappenheim's  method,  51 
Ziehl-Neelsen  method,  51 
in  urine,  168 
typhosus,  417 
in  blood,  244 
technic,  245 
in  urine,  168 
xerosis  in  eye,  382 
Bacteria,  animal  inoculation,  415 
characteristics  of,  415 
collection  of  material  for  cultural 

examination,  412 
cultural  methods  of  examining, 

412 
cultures  of,  study,  413 
Gram-negative,  409 
Gram-positive,  409 
in  blood,  244 
in  feces,  318 

stains  for,  318,  319 
-  in  gastric  contents,  303 
in  milk,  384 
in  pus,  367 
in  sputum,  49 
in  urine,  72,  167 
incubation  of,  413 
inoculating  media  for,  412 


Bacteria,  methods  of  studying,  412 
microscopic  examination,  412 
obtaining  of,   in  preparation  of 

vaccines,  420 
stains  for,  407 

for  morphology,  407 
Bacterial  casts  in  urine,  159 

vaccines,  419.  See  also  Vaccines. 
Bacterins,  419.  See  also  Vaccines. 
Bacteriologic  methods,  396 

study  of  blood,  244 
Bacteriolysis,  265 
Balantidium,  339 

coli,  339 
Basket-cells,  243 

Basophilic   granular  degeneration, 
227 
leukocytes,  240 
structures  of  blood,  220 
Bass    and    Watkins'    method    for 

Widal  reaction,  261 
B.  E.  tuberculin,  428 
Beef  extract  bouillon,  preparation 
of,  401 
infusion,  preparation  of,  401 
tapeworm,  347 
Bence-Jones'  body,  108 

detection,  108 
Benedict's  estimation  of  glucose  in 
urine,  115 
test  for  glucose,  no 
B.  F.  tuberculin,  428 
Bial's  orcin  test  for  pentoses,  118 
Bile  acids  in  urine,  124 
Hay's  test,  124 
tests,  124 
diminished  flow,  indican  in  urine 

from,  91 
in  feces,  315 
in  gastric  contents,  289 
in  urine,  123 
Gmelin's  test  for,  123 
Smith's  test  for,  123 
tests,  123 
medium,  preparation  of,  405 
Bile-pigment  in  urine,  123 
Bilharzia  haematobia,  344 
Bilharziasis,  344 
Bilifuscin  in  urine,  123 
Biliousness,  indican  in  urine  in,  90 
Bilirubin  in  feces,  315 
in  urine,  123 


444 


INDEX 


Biliverdin  in  urine,  123 
Black  sputum,  39 
Bladder,  hemorrhage  from,  166 
schistosomum  haematobium  as 
cause,  167 
Blepharoconjunctivitis,  381 
Blepharoplast  of  tr>panosome,  53:^ 
Blood,  180 

acidophilic  structures  of,  221 
amount  of,  total,  181 
animal  parasites  in,  247 
bacillus  typhosus  in,  244 

technic,  245 
bacteria  in,  244 
bactcriologic  study  of,  244 
basophilic  structures  of,  220 
changes,  albuminuria  from,  loi 
in  blood  diseases,  table,  283 
coagulation  of,  181 
prevention,  182 
time,  181,  182 

Bogg's  method  of  estimating, 
182 
color  index,  200 
color  of,  181 
constituents,  180 
count,  Gibson's  chart  for,   236, 

.237 
diseases,  blood  changes  in,  table, 

283 
Ehrlich's  triple  stain  for,  221 
embryos  of  trichinella  spiralis  in, 

257 
eosin  and  methylene-blue  for,  221 
eosinophilic  structures  of,  221 
erythrocj'tes  in,  number,  192 
filarial  embryos  in,  256 
guaiac  test  for.  274 
Harlow's  stain  for,  224 
hemin  test  for,  274 
in  anaemia  infantum  pseudoleu- 

kaemica,  282 
in  anemia,  276 

aplastic,  279 

pernicious,  277 

secondar>-,  276 
in  aplastic  anemia,  279 
in  chlorosis.  279 
in  feces,  312,  314,  318 
in  gastric  contents,  289,  295,  302 

test  for,  295 
in  Hodgkin's  disease,  282 


Blood  in  leukemia,  280 
in  l>Tnphatic  leukemia,  281 
in  myelogenous  leukemia,  280 
in  pernicious  anemia,  277 
in  pseudoleukemia,  282 
in  splenic  anemia,  280 
in  urine,  72,  166 
increase  of,  184 
leukocytes  in,  number,  202 
malarial  parasites  in,  248.     See 

also  Malarial  parasites. 
neutrophilic  structures  of,  221 
obtaining  of,  for  coagulation  test, 

183 
oxiphilic  structures  of,  221 
parasites,  244 
pathology,  special,  275 
polychrome  methylene-blue  eosin 

stains  for,  222 
reaction  of,  181 
recognition  of,  test  for,  274 
spirochseta  recurrentis  in,  247 
stained,  study  of,  216 
stains,  216 

for  films,  216,  219 
Teichmann's  test  for,  274 
triple  stain  for,  Ehrlich's,  221 
trj'panosoma  gambiense  in,  247 
typhoid  bacilli  in,  244 

technic.  245 
unstained,  malarial  parasites  in, 

253 
volume  index,  200 

Larrabee's  method,  201 
method,  201 
watery,  181 
Wright's  stain  for,  222 
Blood-casts  in  urine,  158 
Blood-corpuscles  in  feces,  318 
red,  180 
decrease    of,    192.      See  also 

Oligocythemia. 
in  gastric  contents,  302 
in  sputum,  63 
in  urine,  165 

increase    of,     192.     See    also 
Polycythemia. 
white,  180 
Blood-dust  of  Miiller,  i8i 
Blood-films,  216 
anilin  dyes  for,  220 
chemic  fixation  of,  219 


INDEX 


445 


Blood-films,  cigarette-paper  meth- 
od for,  218 

drying,  219 

Ehrlich's  two  cover-glass  method, 
217 

fixing,  219 

heat  fixation  for,  219 

Kowarsky's  plate  for  fixing,  219 

making,  216 

malarial  parasites  in,  254 

spreading,  216 

stained,  study  of,  225 

staining,  216,  220 

stains  for,  216,  220 

two-slide  method,  217 
Blood-lancet,  Daland's,  183 
Blood-plaques,  180 

enumeration,  213 

in  anemia,  214 

in  infections,  214 

in  leukemia,  214 

in  purpura  haemorrhagica,  214 

Kemp-Calhoun-Harris      estima- 
tion, 214 

stained,  study  of,  243 

variations  in  numbers,  213 

Wright  and  Kinnicutt's  estima- 
tion, 216 
Blood-platelets,    180.         See   also 

Blood- plaques. 
Blood-serum,  181 

LofHer's  preparation  of,  403 

reactions,  257 
Boas'  reagent,  291 

test  for  free  hydrochloric  acid,  290 

test-breakfast,  286 
Boas-Oppler  bacillus  in  feces,  318 

in  gastric  contents,  303 
Bodies,  Cabot's  ring,  230 

Leishman-Donovan,  335 
Bodo,  336 

urinarius,  336 
Body,  Bence-Jones',  108 

detection,  108 
Boggs'  coagulation  instrument,  182 

method  of  estimating  coagulation 
time  of  blood,  182 

modification  of  Esbach  method 
for  proteins  in  milk,  387 

reagent,  387 

throttle  control  for  blood-count- 
ing pipet,  209,  210 


Boil,  Delhi,  Leishmania  tropica  of, 

335 

Bordet   and   Gengou   test   in   ty- 
phoid fever,  267 

Boston's  method  for  keeping  semen 
for  examination,  391 

Bottles,  vaccine,  420 

Bouillon,  beef  extract,  preparation 
of,  401 
infusion,  preparation  of,  401 

Brick-dust  deposit  in  urine,  72,  143 

Bromids  in  urine,  133 

Bronchi,    cylindric    cells   from,    in 
sputum,  61 

Bronchial  asthma,  eosinophilia  in, 

239 
sputum  in,  66 
Bronchiectasis,  sputum  in,  65 
Bronchitis,  sputum  in,  64 
Brown's  test  for  ammonia  in  urine, 

99 
Bubbles  of  air  in  urine,  173 
Buerger's    method    for    pneumo- 
coccus  capsules,  55 
in  pus,  368 
Butyric  acid  in  gastric  contents,  291 


Cabot's  classification  of  pathologic 
polymorphonuclear      leukocy- 
tosis, 206 
ring  bodies,  230 
Calcium  carbonate  in  iirine,  150 
oxalate  in  urine,  144 
phosphate  crystals  in  urine,  149 
Calculus  in  feces,  313 
renal,  urine  in,  177 
vesical,  urine  in,  178 
Calmette's     ophthalmo-tubercuHn 

reaction,  430 
Cammidge's    pancreatic    reaction, 
129 
technic,  129 
Capsules  of  pneumococcus,  Buer- 
ger's method  for,  54 
Carbol  fuchsin,  50 

thionin  stain  for  bacteria,  408 
Carcinoma,     gastric,    bacteria    in 
feces  in,  318 
gastric  contents  in,  305 
Casts,  fibrinous,  in  sputum,  45 
in  urine,  152 


446 


INDEX 


Casts  in  urine,  negative-staining, 
154 

Catarrh,  vernal,  eosinophilic  leuko- 
cytes in,  383 ,   , 

Cedar  oil  for  oil-immersion  objec- 
tive, 24 

Cells,  alveolar,  in  sputum,  62 
basket-,  243 

body-,  ciliated,  in  sputum,  48 
cylindric,  in  sputum,  61 
eosinophilic,  in  sputum,  60 
epithelial,  in  sputum,  61 

in  urine,  162,  163 
heart-failure,  in  sputum,  41,  62, 

.    65 

in  sputum,  59 
stains  for,  59 

mast-,  240 

polyhedral,  in  urine,  162 

shadow,  in  urine,  166 

squamous,  in  sputum,  61 

vegetable,  in  feces,  316 

yeast-,  in  urine,  171 
Centigrade  and  Fahrenheit  scales, 

440 
Central  illumination  of  microscope, 

19  . 
Centrifuge  for  albumin  in  urine,  106 
for  chlorids  in  urine,  83 

Purdy's  table,  86 
for  sulphates  in  urine,  Purdy's,  89 
Purdy's,  83 
tubes,  Purdy's,  85 
water-motor,  84 
Cercomonas,  335 

hominis,  336 
Cerebrospinal  fever,  epidemic,  cere- 
brospinal fluid  in,  374 
fluid,  bacillus  tuberculosis  in,  374 
examination,  374 
Cestoda,  341,  345 
Cestodes,  341.  345 
Charcot-Leyden  crystals  in  feces, 
320 
in  sputum,  44,  45 
Chart,  Gibson's,  236,  237 
Chemic  examination  of  sputum,  63 

fixation  of  blood-films,  219 
Chemotaxis,  203 
Chlorids  in  urine,  82 

estimation,  Purdy's  centrifugal 
methods,  83 


Chlorids     in     urine,     estimation, 
Purdy's  table,  86 
quantitative,  83,  85 
in  nephritis,  82 
Chlorosis,  279 
color  index  in,  200 
leukopenia  in,  202 
lymphocytes  in,  234 
oligocythemia  in,  193 
Cholesterin  crystals  in  sputum,  45 
Chrysomyia  macellaria,  366 
Chyluria  from  filaria  infection,  148 
Cigarette-paper  method  for  blood- 
films,  218 
Cilia,  328,  339 

Ciliated  body-cells  in  sputum,  48 
Cirrhosis  of  liver,  anemia  from,  276 
Coagulation,  181 

instrument,  Boggs',  182 
prev'ention  of,  182 
time,  181,  182 
Boggs'  method  of  estimating, 
182 
Coccidium,  338 
cuniculi,  338 
Cochin  China  diarrhea,  362 
Coffin-lid  crystals  in  urine,  148 
Colon  bacillus,  417 

in  otitis,  383 
Color  index  of  blood,  200 
in  chlorosis,  200 
in  pernicious  anemia,  200 
of  blood,  181 
Combined  hydrochloric  acid,  284 
Complement,  266,  267,  269 

deviation,  268 
Concretions  in  feces,  313 
Condenser  for  microscope,  22 
Congo-red  test  for  free  acids  in  gas- 
tric contents,  290 
Conjugate  sulphates,  90 
Conjunctivitis,  acute  infectious,  381 
bacteria  of,  381 
blepharo-,  381 
diphtheritic,  382 
gonorrheal,  382 
pseudomembranous,  382 
Cook's  method  for  purin  bodies,  96 
Corpuscles,  blood-,  in  feces,  318 
red,  180 
decrease  of,  192.     See  also 
Oligocythemia. 


INDEX 


447 


Corpuscles,  blood-,  red,  in  gastric 
contents,  302 
in  sputum,  63 
in  urine,  165 

increase  of,  192.      See  also 
Polycythemia. 
white,  180 
pus-,  236,  367 
in  feces,  318 
in  gastric  contents,  302 
in  sputum,  59 
in  urine,  163 
suspension  in  Wassermann  reac- 
tion, 269 
Corrections  for  objectives,  24 
Cotton  fibers  in  urine,  161,  172 
fibrils  in  sputum,  42 
sterilization  of,  400 
Cows'  milk,  384 
Croupous  pneumonia,  sputum  in, 

66 
Cryoscopy  of  urine,  79 
Crystals,  Charcot-Leyden,  in  spu- 
tum, 44,  45 
in  feces,  319 
in  sputum,  45 
Culex,  250 
Cultural    methods    of    examining 

bacteria,  412 
Culture-media,  401 
preparation  of,  401 
reaction  of,  405 
sterilization  of,  399 
tubing,  406 
Cultures  of  bacteria,  study,  413 
Culture-tubes,  397 

preparation  of,  400 
Curds  in  feces,  314 

of  milk  in  feces,  317 
Curschmann's  spirals  in  sputum,  43 
Cyclic  albuminuria,  100 
Cylindric  cells  in  sputum,  61 
Cylindroids  in  urine,  160 
Cysticercus  cellulosse,  348 
Cystin  crystals  in  urine,  146 
Cystinuria,  146 
Cystitis,  urine  in,  178 
Cysts,  daughter-,  349 
Cytodiagnosis,  372 

Daland's  blood-lancet,  183 
hematocrit,  201 


Dare's  estimation  of  hemoglobin, 
189 
hemoglobinometer,  189 

Dark  ground  illumination  of  mi- 
croscope, 21 

Daughter-cysts,  349 

Definitive  host  of  animal  parasites, 

324 
Degeneration  of  Grawitz,  227 
Delhi  boil,  Leishmania  tropica  of, 

335 
Demodex  folliculorum,  366 
Desmoid  test,  Sahli's,  of  gastric  di- 
gestion, 308 
Dextrose  in  urine,   108.     See  also 

Glycosuria. 
Diabetes  insipidus,  urine  in,  179 

mellitus,  urine  in,  1 79 
Diacetic  acid  in  urine,  122 

Gerhardt's  test  for,  122 
Lindemann's  test  for,  122 
tests,  122 
Diarrhea,  Cochin  China,  362 

polycythemia  in,  192 
Diazo  reaction,  126 
in  measles,  128 
in  tuberculosis,  127 
in  typhoid  fever,  127 
technic,  128 
substances  in  urine,  126 
Dibothriocephalus,  351 
latus,  345,  351 
anemia  from,  276 
infection  with,  decrease  of  he- 
moglobin from,  185 
ova  of,  352 
Dicroccelium,  342 
lanceatum,  342 
Diet,  Schmidt's,  for  examination  of 

feces,  320 
Digestion,  gastric,  Sahli's  test  for, 

308 
Digestive  leukocytosis,  205 
Dilatation  of  stomach,  gastric  con- 
tents in,  304 
Diluting  fluids  for  blood  count,  198 
in  leukemia,  213 
for  blood-plaque  count,  215 
Dilution  in  preparation  of  vaccines, 

424 
Diphtheria  bacillus,  417 
of  nasopharynx,  378 


448 


INDEX 


Diphtheria  of  tonsils,  378 
Diphtheritic  conjunctivitis,  382 
Di()lobacillus  of  Morax  and  Axen- 

fcld,  381 
Diplococcus  intracellularis  menin- 
gitidis, 374,  417 

of  Friinkcl  in  sputum,  54 
DipyHdium,  351 

caninum,  351 
Distilling  apparatus,  120 
Dittrich's  plugs  in  sputum,  3Q 
Donne's  test  for  pus  in  urine,  72 
Doremus-IIinds'  ureometer,  94 
Dosage  of  tuberculin,  428 

of  vaccines,  425 

clinical  method,  426 
Dourine,  trypanosoma  equiperdum 

oi,  335 
Drugs,  effect  of,  on  urine,  71,  132 

leukocytosis  from,  207 

resinous,  in  urine,  137 
Drunkard's  pneumonia,  sputum  in, 

39 
Dry  objective,  24 
Dumb-bell   crystals  in   urine,  150, 

151 

Dunham's  peptone  solution,  prep- 
aration of,  404 

Dwarf  tapeworm,  350 

Dysentery,  tropical,  entamoeba  his- 
tolytica in,  328 


Ear,  383 

Earthy  phosphates  in  urine,  87,  149 
Echinococcus  disease,  348 
diagnosis,  349 
eosinophilia  in,  239 
Edema,  pulmonary,  sputum  in,  65 
Eel,  vinegar,  354 

Egyptian  hematuria,  167,  170,  344 
Ehrlich's  diazo  reaction,  126 
technic,  128 
triple  stain  for  blood,  221 
two-cover    method     for    blood- 
films,  217 
Einhorn's  saccharimeter,  114 
Elastic  fibers  in  sputum,  41 
Electric  conductivity  of  urine,  79 
Elephantiasis,  356 
Embryos,  filarial,  in  blood,  256 
filariform,  363 


Embryos  of  trichinella  spiralis  in 
blood, 257 

rhabditiform,  362 
Empty  magnification,  28 
Endocarditis,   malignant,   vaccines 

in,  427 
Entamoeba,  328 

buccalis,  330 

coli,  329 

histolytica,  328 

in  feces,  310 

tetragena,  330 
Enteritis,  membranous,  313 
Enteroliths  in  feces,  313 
Envelope  crystals  in  urine,  144 
Eosin  and  methylene-blue  for  blood, 

221 
Eosinophils,  239 

in  sputum,  60 

in  vernal  catarrh,  383 
Eosinophilia,  239 

in  bronchial  asthma,  239 

in  echinococcus  disease,  239 

in  filariasis,  239 

in  menstruation,  239 

in   myelogenous   leukemia,    239, 
240 

in  parasitic  infections,  183 

in  scarlet  fever,  239,  240 

in  skin  diseases,  239,  240 

in  trichinosis,  239 

in  uncinariasis,  239 

in  worm  infection,  239 
Eosinophilic  cells  in  sputum,  60 

leukocytes,  239 

in  vernal  catarrh,  383 

structures  of  blood,  221 
Epidemic  cerebrospinal  fever,  cere- 
brospinal fluid  in,  374 
Epithelial  casts  in  urine,  158 

cells  in  feces,  317 
in  sputum,  61 
in  urine,  162,  163 
Erythroblasts,  229 
Erythrocytes,  180 

counting  of,  193,  194 

enumeration  of,  192 

in  anemias,  226 

in  gastric  contents,  302 

in  pernicious  anemia,  226 

increase  of,  192 

nucleated,  229 


INDEX 


449 


Erythrocytes,  pessary  forms,  226 

shape  of,  226     , 

size  of,  226 

stained,  study  of,  225 

staining  properties  of,  variations 
in,  226 

structure,  variations  in,  229 

Thoma-Zeiss      instrument      for 
covmting,  193 
Esbach's  albuminometer,  105 

estimation  of  proteins  in  milk, 
Boggs'  modification,  387 

method  for  albumin  in  urine,  105 

reagent  for  albuminuria,  105 
Estivo-autiunnal  parasite,  249,  250, 

256 
Ethereal  sulphates,  90 
Ewald's  salol  test  for  gastric  motor 
power,  307 

test-breakfast,  286 
Exophthalmic  goiter,  lymphocytes 

in,  234 
Exudates,  371 

decomposition     of,     indican     in 
urine  from,  91 
Eye,  381 
Eye-pieces,  microscopic,  23 


Fahrenheit  and  Centigrade  scales, 

440 
False  albuminuria,  100 
Fasciola,  342 

hepatica,  342 
Fat  in  feces,  317 

in  milk,  estimation,  386 
Fat-droplets  in  sputum,  63 

in  urine,  172 
Fat-globules  in  urine,  147 
Fatty  casts  in  urine,  158 
Fatty-acid  crystals  in  sputum,  42 

needles  in  sputum,  45 
Favus,  384 
Feces,  310 

acholic,  312 
-amebae  in,  310 

animal  parasites  in,  313 

bacillus  tuberculosis  in,  319 

bacteria  in,  318 
stains  for,  318,  319 

bile  in,  315 

bilirubin  in,  315 

29 


Feces,  blood  in,  312,  314,  318 
blood-corpuscles  in,  318 
Boas-Oppler  bacillus  in,  318 
calculi  in,  313 
Charcot-Leyden's     crystals    in, 

320 
chemic  examination,  314 
color,  311 
concretions  in,  313 
consistence,  311 
crystals  in,  319 
curds  in,  314 

of  milk  in,  317 
enteroliths  in,  313 
epithelial  cells  in,  317 
erj'throcytes  in,  318 
examination  of,  chemic,  314 

macroscopic,  311 

microscopic,  315 

specimen  for,  310 
fat  in,  317 
flagellates  in,  320 
food  particles  in,  316 
form,  311 

frequency  of  passage,  311 
functional  tests,  320 
Sahli's  glutoid,  321 
Schmidt's  diet,  320 
gall-stones  in,  313 
hydrobilirubin  in,  315 
macroscopic  examination,  311 
maggots  in,  366 
microscopic  examination,  315 
milk  curds  in,  317 
mucus  in,  312 
muscle-fibers  in,  317 
normal,  310 
occult  hemorrhage  in,  detection, 

314 
odor,  312 
ova  in,  320 
parasites  in,  320 
pus  in,  318 
quantity,  311 
starch  granules  in,  316 
tapeworms  in,  313 
trypsin  in,  Miiller's  test  for,  322 
vegetable  cells  in,  316 

fibers  in,  316 
Fehling's  estimation  of  glucose  in 

urine,  114 
test  for  glucose,  no 


450 


INDEX 


Fermentation  method  of  estimating 

glucose  in  urine,  ii6 
Fibers,  elastic,  in  sputum,  41 

in  urine,  extraneous,  161,  172 

muscle-,  in  feces,  317 

of  cotton  in  urine,  161,  172 

of  linen  in  urine,  161,  172 

of  silk  in  urine,  161,  172 

of  wool  in  urine,  161,  172 

vegetable,  in  feces,  316 
Fibrils,  cotton,  in  sputum,  42 
Fibrinous  casts  in  sputum,  45 

in  urine,  157 
F"ilaria,  356 

bancrofti,  356 

diurna,  358 

infection,  chyluria  from,  148 

loa,  358 

medinensis,  358 

perstans,  358 

philippinensis,  358 

sanguinis  hominis,  357 
Filariae  in  urine,  170 
Filarial  embryos  in  blood,  256 
Filariasis,  eosinophilia  in,  239 

parasite  of,  357 
Filariform  embryos,  363 
Fish  tapeworm,  351 
Fixation,  chemic,  for  blood-films, 
219 

heat,  for  blood-films,  219 

of  blood-films,  219 

Kowarsky's  plate  for,  219 
Flagellata,  327,  330 
Flagellates  in  feces,  320 
Flasks,  397 
Flat-worms,  340 
Flaws  in  slides  as  source  of  error, 

173 
Fleischl's  estimation  of  hemoglobin, 

hemoglobinometer,  186 
Flies,  366 

Floaters  in  urine,  169 
Florence's  reaction  for  detection  of 
semen,  392 

reagent,  393 
Flukes,  340,  341 

liver,  342 

lung,  343 
Focal  distance  of  objective,  23,  24 
Focusing  microscope,  29 


Folin's    method    of    quantitative 

estimation  of  urine,  74 
Food  particles  in  feces,  316 

in  gastric  contents,  289,  302 
Formaldehyd  in  milk,  test  for,  388 
Formalin  in  milk,  test  for,  387 
Formalin-gentian- violet  stain,  57 
Friinkel's  diplococcus  in  sputum,  54 
Free    acids    in    gastric    contents, 
Congo-red  test  for,  290 
tests  for,  289 
hydrochloric  acid,  284.    See  also 
Hydrochloric  acid,  free. 
Freezing-point  of  urine,  79 
Friedlander's  bacillus  in  otitis,  383 

in  sputum,  58 
Frommer's  test  for  acetone,  122 
Frothingham's  method  of  demon- 
strating Negri  bodies,  394 
modification    of    van    Gieson's 
stain  for  Negri  bodies,  395 
Fruit-sugar  in  urine,  117 
Functional  tests  for  feces,  320 
Sahli's  glutoid,  321 
Schmidt's  diet,  320 
Fungi,  mold,  in  urine,  172 
Funnel,    separatory,    for    Strauss' 
lactic  acid  test,  293 


Gabbet's  method  for  bacillus  tuber- 
culosis in  sputum,  49 
stain,  51 
Gall-stones  in  feces,  313 
Gametes  in  malaria,  250 
Gangrene  of  lung,  sputum  in,  65 
Gastric  carcinoma,  gastric  contents 
in,  30s 
contents,  acetic  acid  in,  291 
acid  deficit,  299 
bacteria  in,  303 
bile  in,  289 
blood  in,  289,  295,  302 

test  for,  295 
Boas-Oppler  bacillus  in,  303 
butyric  acid  in,  291 
chemic  examination,  289 
constituents,  285 
erythrocytes  in,  302 
examination,  284 
chemic,  289 
microscopic,  301 


See 


Gastric     contents,     examination, 
physical,  288 

routine,  285 
food  particles  in,  289,  302 
free  acids  in,  Congo-red  test 
for,  290 
tests  for,  289 
hydrochloric  acid  in,  284 

also  Hydrochloric  acid. 
in  achylia  gastrica,  305 
in  atrophic  gastritis,  305 
in  carcinoma,  305 
in  dilatation,  304 
in  disease,  304 
in  gastritis,  305 
in  gastrosuccorrhea,  304 
in  neuroses,  304 
in  ulcer,  306 
lactic  acid  in,  291.      See  also 

Lactic  acid. 
leptothrix  buccalis  in,  303 
microscopic  examination,  301 
mucus  in,  288 
obtaining,  285 
organic  acids  in,  291 

quantitative  tests 
pepsin  in,  293 

Hammerschlag's  test 

Mett's  test,  300 

quantitative  test,  299 
Schiitz's,  300 

test  for,  293 
pepsinogen  in,  293 

test  for,  293 
physical  examination,  288 
pus-cells  in,  302 
reaction,  288 

red  blood-corpuscles  in,  302 
rennin  in,  294 

test  for,  294 
renninogen  in,  294 

test  for,  295 
sarcinae  in,  302 
tests,  qualitative,  289 

quantitative,  295 
tissue  bits  in,  289 
total  acidity,  295 
tests,  295 
Topfer's  test,  295 
withdrawal,  287 
yeast-cells  in,  302 
digestion,  Sahli's  test  for,  308 


INDEX  451 

-     .      .  .  ^  ^^TV;.  V 
-GfA^trfc  ^uice,  stimulattonj  ioffifSid- 

8s' 


299 


299 


'  / ;  ,testrn>eals  to  stinlubte/  2/^^  ^  _ 
neuroses,  stomach  contents  m;^  /  H 

ulcer,  gastnc  contents  m,  306         '  l\  , 
Gastritis,    atrophic,    gastric    con- 
tents in,  305 

chronic,  gastric  contents  in,  305 
Gastro-intestinal  diseases,   anemia 

from,  276 
Gastrosuccorrhea,  gastric  contents 

in,  304 
Gauze,  sterilization  of,  400 
Gelatin  media,  sterilization  of,  400 

preparation  of,  402 
Gerhardt's  test  for  diacetic  acid, 

122 
Gibson's  chart,  236,  237 
Giemsa's  stain  for  syphilis,  390 
Glassware,  sterilization  of,  399 
Globular  sputum,  67 
Glossina  palpalis,  248 
Glucose  in  urine,  108.      See  also 

Glycosuria. 
Glutoid  test,  Sahli's,  for  digestive 

functions,  321 
Glycerin  agar-agar,  preparation  of, 

402 
Glycosuria,  108 

Benedict's  quantitative  estima- 
tion, 115 
test  in,  no 

estimation  of  glucose,  112 

Fehling's    quantitative    estima- 
tion, 114 
test  in,  no 

fermentation  method  of  estimat- 
ing, 116 

Haines'  test  in,  109 

Kowarsky's  test  in,  in 

persistent,  109 

phenylhydrazin  test  in,  in 

Purdy's    estimation   of   glucose, 
112 

Robert's      differential      density 
method  of  estimating,  116 

tests,  1 09-11 6 

transitory,  108 
Gmelin's  test  for  bile,  1 23 
Goiter,  exophthalmic,  lymphocytes 

in,  234 


452 


INDEX 


Goiter,  lymphocytes  in,  234 
Gonococcus,  41ft 

in  ci>bthalmia,  382  •  ■  \  ■  ■, 

,  ,        >in  pus,  36g  ' 

in  \irine,  169 
^  ^  v    Gonorrheal  ophthalmia,  382 
threads  in  urine,  169 
CJram-negative  bacteria,  409 
Gram-positive  bacteria,  409 
Gram's  iodin  solution,  57 

method  for  bacillus  influenza  in 
sputum,  58 
for  bacteria  in  feces,  319 
for  pus,  367 
stain  for  bacteria,  409 
Granular  casts  in  urine,  157,  158 

degeneration,  basophilic,  227 
Granule   epithelial   cells   in    urine, 

compound,  162 
Granules,  lycopodium,  in  urine,  173 
starch,  in  feces,  316 
in  urine,  173 
Gravel  in  urine,  142 
Grawitz's  degeneration,  227 
Gray  sputum,  39 
Ground  itch,  361 
Guaiac  test  for  blood,  274 

for  hemoglobin,  125 
Guinea-worm,  358 
Gunning's  test  for  acetone,  1 20 
Gutzeit's  test  for  arsenic  in  urine, 
^33 


Haines'  solution,  no 
test  for  glucose,  109 

Hairs  in  urine,  161 

Hammerschlag's  estimation  of  he- 
moglobin, 189 
test  for  pepsin,  299 

Harlow's  blood  stain,  224 

Hiiser's  method  for  total  solids  in 
urine,  78 

Hayem,  hematoblasts  of,  244 

Hayem's  fluid  for  blood  count,  198 

Hay's  test  for  bile  acids,  1 24 

Heart  disease,  anemia  from,  276 
polycythemia  in,  192 

Heart-failure  cells  in  sputum,  41. 
62,  65 

Heat  and  nitric  acid  test  for  al- 
bumin, 104 


I  Heat  fixation  for  blood-films,  219 
,      test  for  albumin,  Purdy's,  104 

Hematemcsis  and  hemoptysis,  dif- 
j       ferentiation,  289 

Hematoblasts  of  Hayem,  244 

Hematocrit,  193 
Daland's,  201 

Hematoidin    crystals    in    sputum, 

45 
Hematuria,  166 

Egyptian,  167,  170,  344 
from  kidney  tubules,  166 
from  pelvis  of  kidney,  166 
hemoglobinuria  and,  differentia- 
tion, 124 
idiopathic,  166 

Hemin  test  for  blood,  274 

Hemocytometer,  diluting  6uids  for, 
198 
Thoma-Zeiss,  193,  194 
cleaning  instrument,  199 
sources  of  error,  199 
technic,  195 

Hemoglobin,  184 

Dare's  estimation,  189 
decrease  of,  185 
estimation  of,  185-191 
Hammerschlag's  estimation,  189 
in  urine,   124.     See  also  Ilemo- 

globiniiria. 
medium,  preparation  of,  404 
Sahli's  estimation,  187 
Talquist's  estimation,  191 
von  Fleischl's  estimation,  185 

Hemoglobinometer,  Dare's,  189 
Sahli's,  187,  188 
Tallquist's,  190,  191 
von  Fleischl's,  186 

Hemoglobinuria,  124 
detection,  125 
guaiac  test  in,  125 
hematuria    and,    differentiation, 

124 
paroxysmal,  125 
Teichmann's  test  in,  125 
tests,  125 

Hemolysis,  265 

Hemolytic  amboceptor,  269 

Hemoptysis  and  hematemesis,  dif- 
ferentiation, 288 

Hemorrhage,  anemia  from,  276 
from  bladder,  166 


INDEX 


453 


Hemorrhage  from  bladder,  Schisto- 
somum  haematobium  as  cause, 
167 
leukocytosis  after,  207 
occult,  in  feces,  detection,  314 
ELemosporidia,  248 
Herapathite,  137 
Herpetomonas,  335 
Hip-roof  crystals  in  urine,  148 
Hiss'  method  for  pneumococci  in 
pus,  368 
serum  media,  preparation  of,  405 
Hqdgkin's  disease,  282 
Holt's  milk-testing  apparatus,  385 
Hookworm,  anemia  from,  276 
infection,  decrease  of  hemoglobin 
from,  185 
diagnosis,  361 
New  World,  359,  360 
Old  World,  359 
Horismascope,  103 
Host,  definitive,  of   animal    para- 
sites, 324 
intermediate,  of  animal  parasites, 

324 
Hot-air  sterilizer,  396 
Human  milk,  384 
Hyaline  casts  in  urine,  154,  155 
Hydatid  disease,  348 
parasite  of,  349 
Hydrobilirubin  in  feces,  315 
Hydrochloric  acid,  combined,  284 
Topfer's  test  for,  298 
free,  284 
absence,  297 

amidobenzol  test  for,  290 
amount,  297 
decrease,  297 
Boas'  test  for,  290 
increase,  297 
tests  for,  290 

quantitative,  296 
Topfer's  test  for,  297 
Hydrogen  sulphid  generator,  135 
Hydrophobia,    393.  See    also 

-     Rabies. 
HjTTienolepis,  350 

nana,  350 
HjTDerchlorhydria,  297 
H^-perchromemia,  184 
Hyperemia,  active,  urine  in,  173 
passive,  urine  in,  174 


Hyperemia,  renal,  urine  in,  173 
Hyphae  of  molds  in  urine,  161 
Hypobromite  method  for  urea  in 

urine,  94 
Hypochlorhydria,  297 
Hypodermic  injection  of  tubercu- 

Im  for  diagnosis,  429 


Idiopathic  hematuria,  166 

polycythemia,  185,  192 
Illumination,  dark  ground,  of   mi- 
croscope, 21 
for  microscope,  18 

with  water-bottle  condenser,  19 
Immersion  objective,  24 
Immune  bodies,  265,  266 
Incidental  parasites,  340 
Incubation  of  bacteria,  413 
Incubator,  397 
Index,  color,  of  blood,  200 
opsonic,  263 
phagocytic,  263 
volume,  of  blood,  200 

Larrabee's  method,  201 
method,  201 
India-ink  method  for  syphilis,  391 
Indican  in  urine,  91 
detection,  91 

from   decomposition    of    exu- 
dates, 91 
from  diminished  flow  of  bile,  91 
in  biliousness,  90 
in  diseases  of  small  intestine,  90 

of  stomach,  90 
Obermayer's  test  for,  91 
tests  for,  91 
Indicanuria,  90.     See  also  Indican 

in  urine. 
Infection,  blood-plaques  in,  214 
leukocytosis  in,  206 
mixed,  54 

phagocytosis  and,  261 
vaccines  in,  426,  427 
Infecrious  diseases,  secondary  ane- 
mia from,  276 
Inflammations,  leukocytosis  in,  206 
pseudomembranous,    of    mouth, 
378 
Influenza  bacillus,  418 
in  spinal  fluid,  376 
in  sputum,  58 


454 


INDEX 


Infusion,  beef,  preparation  of,  401 
bouillon,  preparation  of,  401 

Infusoria,  328,  339 

Inoculating  media  for  bacteria,  412 

Inoculation,  animal,  375 
of  bacteria,  415 

Intermediate  host  of  animal  para- 
sites, 324 

Intestine,  small,  diseases  of,  indican 
in  urine  in,  90 

Intoxication,  acid,  cause,  118 

lodin  in  urine,  133 

reaction  of  leukocytes,  238 
solution.  Gram's,  57 

Iodoform  crystals  from  Gunning's 
test,  121 

lodophilia,  238 

Irregular  malaria,  249 

Itch,  ground,  361 
mite,  366 


Kala-azar,  Leishmania  donovani 

of,  335 

Kelling's  test  for  lactic  acid,  292 

Kemp-Calhoun-Harris     estimation 
of  blood-plaques,  214 

Kidney,    changes   in,   albuminuria 
from,  loi 
permeability  of,  tests  for,  78,  79 

Koch-Weeks  bacillus  in  conjuncti- 
vitis, 381 

Kowarsky's   plate   for   fixation   of 
blood-films,  219 
test  for  glucose,  11 1 


Lactic  acid  in  gastric  contents,  291 
Kelling's  test  for,  292 
Simon's  test  for,  292 
Strauss'  test  for,  292 
Uffelmann's  test  for,  292 
Lactose  in  milk,  estimation,  387 

in  urine,  117 
Lamblia,  338 

intestinalis,  338 
Lamp,  Matthews'  microscope,  19, 

20 
Lancet,  blood-,  183 
Lange's  test  for  acetone,  121 
Larrabee's    estimation    of    volume 
index  of  blood,  201 


Lead  in  urine,  134 

Lederer's  test,  134 
Lead-poisoning,  anemia  from,  276 

chronic,  degeneration  of  Grawitz 
in,  228 
Lederer's  test  for  lead  in  urine,  134 
Lefifmann-Beam  estimation  of  fat 

in  milk,  386 
Legal 's  test  for  acetone,  Lange's 

modification,  121 
Leishman-Donovan  bodies,  335 
Leishmania,  335 

donovani,  335 

infantum,  335 

tropica,  335 
Leishman's  method  for  measuring 

opsonins,  263 
Lenses,  23 

for  microscope,  care  of,  30 
Leprosy,  secondary  anemia  from, 

276 
Leptothrix  buccalis,  377 
in  gastric  contents,  303 
in  sputum,  42 
Leucin  in  urine,  145 
Leukemia,  203,  208,  280 

blood-plaques  in,  214 

Boggs'  estimation  of  leukocytes 
in,  209 

degeneration  of  Grawitz  in,  228 

diluting  fluids  for  count,  213 
Leukemia,  eosinophilia  in,  239,  240 

erythroblasts  in,  230 

leukocyte  count  in,  209 

lymphatic,  281 

lymphocytes  in,  234 

mast-cells  in,  241 

myelocytes  in,  242 

myelogenous,  280 

oligocythemia  in,  193 

polychromatophilia  in,  227 

Todd's  estimation  of  leukocytes 
in,  211 

Turck's  ruling  for  blood  count  in, 
209,  211 

Zappert  ruling  for  blood  count 
in,  209 
Leukocytes,  180 

abnormal  varieties,  241 

atypic  forms,  242 

basophilic,  240 

border-line  forms,  243 


INDEX 


455 


Leukocytes,  classification  of,  231 
counting,  in  leukemia,  209 
decrease  in,  201 
degenerated  forms,  243 
differential  count  of,  230 
enumeration,  202 
eosinophilic,    239.  See    also 

Eosinopkiles. 
increase  in,  203 

absolute,  231 

relative,  231 
iodin  reaction  of,  238 
irritation  forms,  243 
mononuclear,  large,  234 
normal,  232 
polymorphonuclear,  neutrophilic, 

235 
polynuclear,  236 
stained,  study  of,  230 
transitional,  235 
vacuolated,  243 
Leukocytosis,  203 
absolute,  231 
digestive,  205 
lymphocyte,  204,  207 

in  hereditary  syphilis,  208 
in  pertussis,  208 
myelocytes  in,  242 
non-phagocytic,  208 
permanent,  203 
polymorphonuclear,  204 
from  drugs,  207 
from  infections,  206 
from  inflammations,  206 
in  malignant  disease,  207 
pathologic,  205 
physiologic,  205 
toxic,  207 
relative,  231 
transient,  203 
Leukopenia,  202 
in  chlorosis,  202 
in  pernicious  anemia,  202 
lymphocytes  in,  233 
Levulose  in  urine,  117 
Lieben's  test  for  acetone.  Gunning's 

modification,  121 
Linen  fibers  in  urine,  161,  172 
Litmus  milk,  preparation  of,  404 
Liver,  cirrhosis  of,  anemia  from,  276 
fluke,  342 
rot,  342 


Loffler's   alkaline    methylene-blue, 

57 
blood-senmi,  preparation  of,  403 
methylene-blue  for  gonococci  in 
pus,  369 
for  pus,  367 
stain  for  flagella,  411 
Louse,  366 

Lung,  edema  of,  sputum  in,  65 
fluke,  343 

gangrene  of,  sputum  in,  65 
tuberculosis  of,  sputiun  in,  66 
Lycopodium  granules  in  urine,  173 

used  as  micrometer,  33 
LjTnphatic  leukemia,  281 
Ijrmphocytes  in,  234 
Lymphocyte  leukocytosis,  204,  207 
in  hereditary  syphilis,  208 
in  pertussis,  208 
Lymphocytes,  232 
Lymphocytosis,  208 


Macrocytes,  226 

Macroscopic  examination  of  spu- 
tum, 37 

Maggots  in  feces,  366 

Magnification,  empty,  28 
microscopic,  27 
methods  of  increasing,  28 

Malaria,  irregular,  249 
large  mononuclear  leukocytes  in, 

234 
parasites   of,    248.         See   also 

Malarial  parasites. 
secondary  anemia  from,  276 
transmission   of,   by   mosquitos, 

250 
Malarial  parasites,  248 

ameboid  movements  of,  248 

cycles  of,  248 

asexual  cycle,  248 

detection,  251,  253,  254 

estivo-autumnal,  249,  250,  256 

gametes  in  blood  with,  250 

hyaline  stage  of,  248 

life  histories,  248 

merozoites  of,  248 

mosquitos  as  host,  250 

quartan,  250,  256 

Ruge's  stain  for,  254 

segmentation  of,  248 


456 


INDEX 


Malarial    parasites,  sexual   cycle, 
248,  249 
spores  of,  248 
stains  for,  254 
tertian,  249,  250,  256 
Wright's  stain  for,  254 
stippling,  228 
Malignant  disease,  leukocytosis  in, 
207 
endocarditis,  vaccines  in,  427 
tumors,  anemia  from,  276 
Mast-cells,  240 
Mastigophora,  327,  330 
Matthews'  microscope  lamp,  19.  20 
McFarland's    method    for    Widal 

reaction,  259 
Measles,  diazo  reaction  in,  128 
Measures,  439 

Media,   culture-,  401.        See  also 
Culture-media. 
gelatin,  sterilization  of,  400 
Megaloblasts,  229 
Megalocytes,  226 
Melanin  in  urine,  126 

tests  for,  126 
Melanogen  in  urine,  126 
^lelanuria,  126 
Membranous  enteritis,  313 
Meningitis,    tuberculous,    cerebro- 
spinal fluid  in,  374 
Menstruation,  eosinophilia  during, 

239 
Mercury  in  urine,  136 

treatment  of  syphilis,  effect  of, 
on  Wassermann  reaction,  273 
Merozoites   of   malarial   parasites. 

248 
Metal,  sterilization  of,  399 
Methylene-blue  eosin  stains,  poly- 
chrome, for  blood,  222 

test  for  urine,  79 
Metric  system,  439 
Mett's  test  for  pepsin,  300 

tubes,  399 
Microblasts,  229 

Micrococcus  catarrhalis  in  sputum, 
58.  59 

urae  in  urine,  167 
Microcytes,  226 

Micrometer    eye-piece    for   micro- 
scope, 31,  32 

stage,  32 


Micron,  32 
Microscope,  17 

care  of,  30 

choice  of,  31 

cleaning,  30 

condenser  for,  22 

eye-pieces  for,  23 

focusing,  28 

illumination  for,  18 
dark  ground,  21 

lamp,  Aiatthews,  19,  20 

lenses  for,  23 
care  of,  30 

magnification  by,  27 

methods  of  increasing,  28 

method  of  carrying,  30 

micrometer  eye-piece  for,  31,  32 

objectives  for,  23 
corrections,  2*4 

use,  17 
^licroscopic  objects,  measurement, 

31 

Micturition,  frequency  of,  70 

Milk,  384 

analysis  of,  384 
tube  for,  386 
bacteria  in,  384 
chemic  examination,  385 
curds  of,  in  feces,  317 
fat  in,  estimation,  386 
formalin  in,  test  for,  387 
lactose  in,  estimation,  387 
litmus,  preparation  of,  404 
proteins  in,  estimation,  387 
reaction,  384 

Milk-sugar  in  urine,  117 

Milk-testing  apparatus.  Holt's,  385 

Mineral  sulphates,  90 

Mite,  itch,  366 

Mixed  infection,  54 

Moeller's  stain  for  spores,  410 

Mold  fungi  in  urine,  172 

Molds,  hj-pha;  of,  in  urine,  161 
in  sputum,  47 

Mononuclear  leukoc>'tes,  large,  234 

Morax  and  Axenfeld's  diplobacil- 
lus,  381 

Moro's  tuberculin  reaction  in  diag- 
nosis of  tuberculosis,  430 

Morphin  in  urine,  136 

Mosquitos  in  transmission  of  mala- 
ria, 250 


INDEX 


457 


Motor  power  of  stomach,  307 
Mouth,  diseases  of,  377 

organism  of,  377 
Mucin  in  urine,  106 
Mucous  threads  in  urine,  159 
Mucus  in  feces,  312 
in  gastric  contents,  288 
in  urine,  159 
Miiller's  blood-dust,  181 
fluid,  56 

test  for  trypsin  in  feces,  322 
Muscle-fibers  in  feces,  317 
Myelin  globules  in  sputum,  63 
Myelocytes,  241 
Myelogenous  leukemia,  280 
eosinophilia  in,  239,  240 
erythroblasts  in,  230 
mast-cells  in,  241 
myelocytes  in,  242 

Nagana,  trypanosoma  brucei  of, 

335 
Nasopharynx,  diphtheria  of,  378 
Necator  americanus,  359,  360 

life-history,  360 
Needles,  fatty-acid,  in  sputum,  45 
Negative-staining  of  urinary  casts, 

154 
Negri  bodies,  393 

Frothingham's  method  of  de- 
monstrating, 394 
Van  Gieson's  stain  for,  Froth- 
ingham's modification,  395 
Nemathelminthes,  341,  353 
Nematoda,  353 
Nematodes,  353 
Nephritis,  anemia  from,  276 

chlorids  in  urine  in,  82 

urine  in,  173,  175,  176 
Neuroses,  gastric,  stomach  contents 

in,  304 
Neutrophilic  leukocytes,  polymor- 
phonuclear, 235 

structures  of  blood,  221 
Newton's  rings,  196 
Nitric  acid  test  for  albumin,  104 
Nitrogen  equilibrium,  92 

partition,  92 
Noguchi  test  for  syphilis,  271 
Normoblasts,  229 
Nose,  cylindric  cells  from,  in  spu- 

timi,  6i 


Nubecula  of  urine,  72 
Numeric  aperture,  25 
Nutrition,  poor,  secondary  anemia 
from,  276 


Obermayer's  reagent,  91 

test  for  indican  in  urine,  91 
Objectives,  achromatic,  23 

apochromatic,  23 

dry,  24 

focal  distance  of,  23,  24 

immersion,  24 

microscopic,  23 

niuneric  apertures,  25 

oil-immersion,  24,  25 

resolving  power  of,  25 

working  distance  of,  23 
Oblique  illumination  of  microscope, 

20 
Occult  hemorrhage  in  feces,  detec- 
tion, 314 
O'idlum  albicans,  378 
Oil-immersion  objective,  24,  25 
Oligochromemia,  185 
Oligocythemia,  192 

in  anemias,  193 

in  chlorosis,  193 

in  leukemia,  193 
Oliguria,  71 
Oncospheres,  346 
Ophthalmia,  gonorrheal,  382 
Ophthalmo-tuberculin        reaction, 

Calmette's,  430 
Opisthorchis,  343 

felineus,  343 

sinensis,  343 
Opsonic  index,  263 
Opsonins,  261 

Leishman's  method  for  measur- 
ing, 263 

measuring  amount  of,  262 

Wright's  method  for  measuring, 
262 
Orcin  test,  Bial's,  for  pentoses,  118 
Organic  acids  in  gastric  contents, 
291 
quantitative  tests,  299 
Oriental  sore,  Leishmania  tropica 

of,  235 
Orthostatic  albuminuria,  100 
Otitis,  383 


458 


INDEX 


Otitis,  bacteria  of,  383 

tuberculous,  383 
Ova  in  feces,  320 
Oxybutyric  acid  in  urine,  123 
Oxj^ihilic  structures  of  blood,  221 
Oxyuris,  355 

vermicularis,  355 
ova  of,  356 


Pancreatic  reaction,  129 

flasks  for,  130 

in  pancreatitis,  129 

technic,  129 
Pancreatitis,  pancreatic  reaction  in, 

129 
Pappenheim's  method  for  bacillus 

tuberculosis  in  sputum,  51 
pyronin-methyl-green    for    bac- 
teria, 408 

for  gonococci  in  pus,  369 

for  pus,  367 
Paragonimus,  343 
westermani,  343 

in  sputum,  48 
Paramoecium  coli,  339 
Parasites,  animal,  323 

anemia  from,  276 

arthroiX)da,  366 

classification,  324,  325 

definitive  host,  324 

in  blood,  247 

in  feces,  313 

in  sputum,  48 

in  urine,  169 

infection  with,  eosinophilia  in, 

239 
intermediate  host,  324 
nomenclature,  324,  325 
protozoa,  326,  327 
blood,  244 

causing  skin  diseases,  384 
in  feces,  320 
incidental,  340 

malarial,  248.    See  also  Malarial 
parasites. 
Paroxysmal  hemoglobinuria,  125 
Pavement  epithelial  cells  in  urine, 

163 
Pediculus  capitis,  366 
pubis,  366 
vestimenti,  366 


Pemphigus,  eosinophilia  in,  240 
Pentoses  in  urine,  117 

Bial's  orcin  test,  118 
Pepsin  in  gastric  contents,  293 

Hammerschlag's  test,  299 
Mett's  test,  300 
quantitative  test,  299 
Schutz's  law,  300 
test  for,  293 
Pepsinogen  in  gastric  contents,  293 

test  for,  293 
Peptone  solution,  Dunham's,  prep- 
aration of,  404 
Pericardial  fluid,  examination,  371 
Peritoneal  fluid,  examination,  371 
Permeability  of  kidneys,  test,  78,  79 
Pernicious  anemia,  277 
blood-plaques  in,  214 
color  index  in,  200 
degeneration  of  Grawitz  in,  228 
erythroblasts  in  blood  in,  230 
erythrocytes  in,  226 
leukopenia  in,  202 
lymphocytes  in,  234 
myelocytes  in,  242 
polychromatophilia  in,  227 
Pertussis,  lymphocyte  leukocytosis 
in,  208 
lymphocytes  in,  234 
Pessary  forms  of  erythrocytes,  226 
Pfeiffer's  phenomenon,  265 
Phagocytic  index,  263 
Phagocytosis,  206 

and  infection,  261 
Pharyngomycosis  leptothrica,  377 
Pharynx,  tuberculosis  of,  380 

ulceration  of,  380 
Phenacetin  in  urine,  132 
Phenol  in  urine,  136 
Phenolphthalein  in  urine,  137. 
Phenylglucosazone  crystals,   no 
Phenylhydrazin    test   for   glucose, 

III 
Phloridzin  test  for  urine,  79 . 
Phosphate  crystals  in  urine,  am- 
moniomagnesium,   148 
calcium,  149 
triple,  148 
Phosphates  in  urine,  86,  148 
alkaline,  87 

amorphous,  72,  87,  149 
in  mass,  160 


INDEX 


459 


Phosphates  in  urine,  decreased,  87 
earthy,  87,  149 
estimation,  87,  88 
Purdy's  centrifuge  method, 

88 
quantitative,  87 
increased,  87 
Purdy's  table  for,  after  cen- 

trifugation,  88 
triple,  87 
Phosphaturia,  87 
Phosphorus-poisoning,  anemia 

from,  276 
Photomicrography,  S3 
Physiologic  albuminuria,  100 
Pink-eye,  381 
Pin- worm,  355 
Pipets,  398 

for  counting  vaccines  by  Wright's 
method,  422 
Piroplasma  hominis,  339 
Pirquet's  reaction  in  tuberculosis, 

430 
Plasmodium,  338,  339 

falciparum,  248 

malariae,  248.     See  also  Malarial 
parasites. 

vivax,  248 
Platinum  wires,  397 
Platyhelminthes,  340,  341 
Pleural  fluid,  examination,  371 
Plugs,  Dittrich's,  in  sputum,  39 
Pneumococcus,  416 

capsules,  Buerger's  method  for, 

55 
in  eye  affections,  381 

otitis,  383 
in  pus,  368 
sputum,  54 
Smith's  method,  56 
Pneumonia,  croupous,  sputum  in, 
66 
drunkard's,  sputum  in,  39 
Poikilocytes,  226 
Poikilocytosis,  226 
Poisoning,   arsenic,   anemia   from, 
276 
lead-,  anemia  from,  276 
phosphorus-,  anemia  from,  276 
Polychromatophilia ,  227 
Polychrome   methylene-blue   eosin 
stains  for  blood,  222 


Polycythemia,  192 

idiopathic,  185,  192 

in  diarrhea,  192 

in  heart  disease,  192 
Polyhedral  cells  in  urine,  162 
Polymorphonuclear      leukocytosis, 

204,  205.     See  also  Leukocytosis, 
polymorphonuclear. 

neutrophilic  leukocytes,  235 
Polynuclear  leukocytes,  236 
Polyuria,  70 
Pork  tapeworm,  348 
Posthemorrhagic  leukocytosis,  207 
Postural  albuminuria,  100 
Potassium     indoxyl     sulphate     in 

urine,  90.      See  also  Indican  in 

urine. 
Potato  medium,  preparation  of,  404 
Power  of  resistance,  236 
Preformed  sulphates,  90 
Pregnancy,  urine  in,  175 
Primary  proteoses  in  urine,  106 
Proglottides,  345 

Progressive  pernicious  anemia,  277 
Proteins  in  milk,  estimation,  387 

in  urine,  99 
Proteoses  in  urine,  106 
detection,  108 
primary,  106 
secondary,  106 
Protozoa,  326,  327 
Prune-juice  sputum,  39 
Prurigo,  eosinophilia  in,  240 
Pseudocasts  in  urine,  161 
Pseudoleukemia,  282 
Pseudomembranous    conjunctivits, 
382 

inflammations  of  mouth,  378 
Psoriasis,  eosinophilia  in,  240 
Pulmonary  edema,  sputum  in,  65 

gangrene,  sputum  in,  65 

tuberculosis,  sputum  in,  66 
tuberculin  in,  428,  249 
Purdy's   centrifugal   estimation  of 
albumin,  106 
of  chlorids,  83 
of  phosphates,  88 
of  sulphates,  89 

centrifuge  tubes,  85 

electric  centrifuge,  83 

estimation  of  glucose  in  urine,  112 

heat  test  for  albumin,  104 


460 


INDEX 


Purdy's  solution  for  glucose  test, 

table  for  estimation  of  albumin, 
107 
of  chlorids,  86 
of  phosphates,  88 
of  sulphates,  89 
Purin  bodies  in  urine,  95 

Cook's  method,  96 
Purpura      haemorrhagica,      blood- 
plaques  in,  214 
Pus,  bacillus  tuberculosis  in,  369 
bacteria  in,  367 
examination  of.  367 
gonococci  in,  369 
Gram's  method  for,  367 
in  feces,  318 
in  urine,  72,  164 

Donne's  test,  72 
Lofller's  methylene-blue  for,  367 
Pappenheim's     pyronin-methyl- 

green  for,  367 
pneumococci  in,  368 
staphylococci  in,  368 
streptococci  in,  368 
Pus-casts  in  urine,  159 
Pus-corpuscles,  236,  367 
in  feces,  318 
in  gastric  contents,  302 
in  sputum,  59 
in  urine,  163 
Pyelitis,  urine  in,  177 
Pyuria,  164 


Quartan  parasite,  250,  256 
Quinin  in  urine,  137 


Rabies,  diagnosis  of,  393 

Frothingham's    method    of    de- 
monstrating Negri  bodies  in, 

3Q4 
Ray-fungus  in  sputum,  46 
Reaction,  Noguchi's,   for  syphilis, 

271 
Wassermann,    274.         See   also 

Wasserniann  reaction. 
Reagents.  434 
Red  blocxi-corpuscles,  180 

decrease  of,   192.        See  also 
Oligocythemia. 


Red    blood-corpuscles    in    gastric 
contents,  302 
in  sputum,  63 
in  urine,  165 

increase   of,    192.        See  also 
Polycytticmia. 
sand  in  urine,  142 
Reinsch's  test  for  arsenic  in  urine, 

133 
Relapsing  fever,  spirochaeta  of,  247, 

331 
Renal  albuminuria,  100 

calculus,  urine  in,  177 

circulation,    changes    in,    albu- 
minuria from,  loi 

hyperemia,  urine  in,  173 

tuberculosis,  urine  in,  175 
Rennin  in  gastric  contents,  294 

test  for,  294 
Renninogcn  in  gastric  contents,  294 

test  for,  295 
Resinous  drugs  in  urine,  137 
Resistance,  power  of,  236 
Resolving  jx)\ver  of  objective,  25 
Rhabditiform  embryos,  362 
Rheumatism,      secondary  anemia 

from,  276 
Rhizojxxla,  327,  328 
Rice's  solutions,  95 
Ring  bodies,  Cabot's,  230 
Rings,  Xewton's,  196 
Ringworm.  384 

Robert's  differential  density  meth- 
od of  estimating  glucose  in 
urine,  116 

test  for  albumin,  103 
Ronchese-Malfatti  formalin  test  for 

ammonia  in  urine,  98 
Round- worms,  354 

in  children,  354 
Ruge's  stain  for  malarial  parasites, 

254 
Ruhemann's  method  for  uric  add, 

97 
reagent,  97 
uricometer,  96 
Rusty  sputum,  38,  39 


Saccharimeter,  Einhom's,  1 14 
Sahli's  desmoid  test  of  gastric  di- 
gestion, 308 


INDEX 


461 


Sahli's  estimation  of  hemoglobin, 
187 

glutoid  test  for  digestive  func- 
tions, 321 

hemoglobinometer,  187,  188 
Salicylates  in  urine,  137 
Salol  in  urine,  137 

test,  Ewald's,  for  gastric  motor 
power,  307 
Salvarsan    treatment    of    syphilis, 

effect  of,  on  Wassermann  reac- 
tion, 273 
Sand,  red,  in  urine,  142 
Sarcinae  in  gastric  contents,  302 
Sarcodina,  327,  328 
Sarcoptes  scabiei,  366 
Saxe's  urinopyknometer,  76 
Scarlet  fever,  eosinophilia  in,  239, 

240 
Schistosomum,  344 

hematobium,  342,  344 
in  urine,  170 

in  veins  of  bladder  as  cause  of 
hemorrhage,  167 

japonicum,  345 
Schmidt's  diet  for  examination  of 

feces,  320 
Schiitz's  law  in  quantitative  test  for 

pepsin,  300 
Scolex,  345 
Scratches    on    slide    as    source    of 

error,  173 
Screw  worm,  366 
Secondary  anemia,  276 

proteoses  in  urine,  106 
Secretory  ability  of  kidneys,  tests, 

78,  79 
Sediments,  urinary,  138.     See  also 

Urinary  sediment. 
Segmentation  of  malarial  parasites, 

248 
Semen,  examination  of,  391 

on  clothes,  detection,  392 
Florence's  reaction  for,  392 
Separatory    funnel     for     Strauss' 

lactic  acid  test,  293 
Scrum,   blood-,   Loffler's,  prepara- 
tion of,  403 

media,  Hiss',  preparation  of,  405 

reactions,  257 
Serum-albumin  in  urine,  99 
Serum-globulin  in  urine,  99 


Shadow  cells  in  urine,  166 
Silk  fibers  in  urine,  161,  172 
Silver    impregnation    method    for 
syphilis,  390 

nitrate  solution,  ammoniated,  97 
Simon's  test  for  lactic  acid,  292 
Skin  diseases,  eosinophilia  in,  239, 
240 

parasitic  diseases  of,  384 
Sleeping  sickness,  248 

trypanosoma  gambiense  of,  334 
Small  intestine,  diseases  of,  indican 

in  urine  in,  90 
Smegma  bacillus,  53,  169 
Smith's  method  for  pneumococcus 
in  sputum,  56 

test  for  bile,  123 
Sodium  urate  in  urine,  144 
Specific  gravity  of  urine,  74 
Spermatozoa,  absence  of,  391 

in  urine,  167 
Spinal  fluid,  influenza  bacilli  in,  376 
Spirals,  Curschmann's,  in  sputum, 

43 
Spirochaeta,  330 

buccalis,  332 

carter i,  331 

dentium,  332 

duttoni,  331 

kochi,  331 

novyi,  331 

obermeieri,  331 

pallida,  388 

recurrentis,  330 
in  blood,  247 

refringens,  2>3,3 
in  syphilis,  389 

vincenti,  331,  380 
Splenic  anemia,  280 
Splenomegaly,      infantile,      Leish- 

mania  infantum  of,  335 
Spores  of  malarial  parasites,  248 
Sporozoa,  328,  338 
Sputum,  36 

actinomyces  bovis  in,  46 

albumin  in,  63 

alveolar  cells  in,  62 

Amceba  histolytica  in,  48 

animal  parasites  in,  48 

bacillus  mucosus  capsulatus  in, 
58 
of  Friedlander  in,  58 


462 


INDEX 


Sputum,  bacillus  of  influenza  in,  58 
tuberculosis  in,  36,  49 

bacteria  in,  49 

black,  39 

cells  in,  59 
stains  for,  59 

Charcot-Leyden    crystals  in,  44, 

45 
chemic  examination,  63 
cholesterin  crystals  in,  45 
ciliated  body-cells  in,  48 
collection  of,  36,  37 
color  of,  38 
consistence,  39 
cotton  fibrils  in,  42 
crudum,  39 
crystals  in,  44,  45 
Curschmann's  spirals  in,  43 
cylindric  cells  in,  61 
diplococcus  of  Frankel  in,  54 
Dittrich's  plugs  in,  39 
elastic  fibers  in,  41 
eosinophilic  cells  in,  60 
epithelial  cells  in,  61 
examination,  36 

chemic,  63 

macroscopic,  37 

microscopic,  40 

physical,  38 
fat-droplets  in,  63 
fatty-acid  crystals  in,  42 

needles  in,  45 
fibrinous  casts  in,  45 
Frankel's  diplococcus  in,  54 
globular,  67 
gray,  39 

heart-failure  cells  in,  41,  62,  65 
hematoidin  crystals  in,  45 
in  bronchial  asthma,  66 
in  bronchiectasis,  65 
in  bronchitis,  64 
in  croupous  pneumonia,  66 
in  disease,  64 

in  drunkard's  pneumonia.  39 
in  gangrene  of  lung,  65 
in  pneumonia,  croupous,  66 
in  pulmonary  edema,  65 

gangrene,  65 

tuberculosis,  66 
leptothrix  buccalis  in,  42 
macroscopic  examination,  37 
micrococcus  catarrhalis  in,  58,  59 


Sputum,  molds  in,  47 

myelin  globules  in,  63 

Paragonimus  westermani  in,  48 

pneumococcus  in,  54 
Smith's  method,  56 

prune-juice,  39 

pus-corpuscles  in,  59 

quantity,  38 

ray-fungus  in,  46 

receptacle  for,  37 

rusty,  38,  39 

squamous  cells  in,  61 

stained,  48 

staphylococci  in,  54 

streptococci  in,  54 

streptothrix  actinomyces  in,  47 

tubercle  bacillus  in,  36,  49 

unstained,  40 

yeasts  in,  47 
Squamous  cells  in  sputum,  61 

epithelial  cells  in  urine,  163 
Squibb's  urinometer,  75 
Stage  micrometer,  32 
Stained  blood,  216 

sputum,  48 
Staining  methods,  407 
Stains,  434 

anilin,  for  blood- films,  220 

anilin-gentian  violet,  57 

carbol  fuchsin,  50 

thionin,  for  bacteria,  408 

Ehrlich's  triple,  for  blood,  221 

eosin    and    methylene-blue,   for 
blood,  221 

for  bacillus  influenza  in  sputum, 

tuberculosis  in  sputum,  49 
for  bacteria,  407 

in  feces,  318 

in  sputum,  49 

for  morphology,  407 
for  blood,  216 

Wright's,  in  cytodiagnosis,  372 
for  blood-films,  216,  220 
for  cells  in  sputum,  59 
for  malarial  parasites,  254 
for  pneumococcus  capsules,  55 
for  pus,  367 
for  syphilis,  390 
formalin-gentian-violet,  57 
Gabbet's,  51 
Giemsa's,  for  syphilis,  390 


INDEX 


463 


Stains,  Gram's,  for  bacteria,  409 

for  pus,  367 

iodin  solution,  57 
Harlow's,  for  blood,  224 
India-ink,  for  syphilis,  391 
iodin,  for  leukocytes,  238 
iodin  solution.  Gram's,  57 
LoflBer's  alkaline  methylene-blue, 

57 
for  flagella,  411 

methylene-blue,  for  gonococci 
in  pus,  369 
for  pus,  367 
Moeller's,  for  spores,  410 
negative-,  for  urinary  casts,  154 
Pappenheim's     pyronin-methyl- 
green,  367 
for  bacteria,  408 
for  gonococci  in  pus,  369 
polychrome  methylene-blue  eosin , 

for  blood,  222 
Ruge's,  for  malarial  parasites,  254 
silver,  for  syphilis,  390 
Stirling's      anilin-gentian-violet, 

57 
triple,  for  blood,  221 
Van  Gieson's,  for  Negri  bodies, 

Frothingham's      modification, 

395 
Wright's,  for  blood,  222 
in  cytodiagnosis,  372 
for  malarial  parasites,  254 
for  syphilis,  390 
Staphylococci,  368 
in  eye  affections,  381 
in  otitis,  383 
in  sputum,  54 
Staphylococcus  pyogenes  albus,  416 
aureus,  415 
citreus,  416 
Starch  paper,  307 
Starch-granules  in  feces,  316 

in  urine,  173 
Steam  sterilizer,  396 
Sterility,  391 
Sterilization,  399 

in  preparation  of  vaccines,  422 

of  cotton,  400 

of  culture-media,  399 

of  gauze,  400 

of  gelatin  media,  400 

of  glassware,  399 


Sterilization  of  metal,  399 
Sterilizers,  396 

dry,  396 

hot-air,  396 

steam,  396 
Stippling,  malarial,  228 
Stirling's  anilin-gentian-violet  stain, 

57 
Stock  vaccines,  419 
Stomach,  284 

absorptive  power  of,  306 

contents  of,  284.    See  also  Gastric 
contents. 

digestion,  284 

dilatation  of,  gastric  contents  in, 

.304 
diseases  of,  indican  in  urine  in,  90 
motor  power  of,  307 
position  of,  determination,  308 
size  of,  determination,  308 
Stomach- tube,  287,  288 
Stools,  310.     See  also  Feces. 
Strauss'  test  for  lactic  acid,  292 
Streptococci,  368 
in  eye  affections,  381,  382 
in  otitis,  383 
in  sputum,  54 
Streptococcus  pyogenes,  416 
Streptothrix  actinomyces  in  spu- 
tum, 47 
Strongyloides,  362 

intestinalis,  324,  362,  363 
Sugar  media,  preparation  of,  403 
Sugars  in  urine,  108 
Sulphates,  conjugate,  90 
ethereal,  90 
in  urine,  88 
estimation,  Purdy's  centrifugal 
method,  89 
quantitative,  89 
Purdy's  table  after  centrifuga- 
tion,  89 
mineral,  90 
preformed,  90 
Sulphuric  acid  in  urine,  88 
Surra,  trypanosoma  evansi  of,  335 
Syphilis,  dark  ground  illumination 
in,  391 
examination  of  material,  388 
Giemsa's  stain  for,  390 
hereditary,    lymphocyte    leuko- 
cytosis in,  208 


464 


INDEX 


Syphilis,  India-ink  method  for,  391 
micro-organism  of,  388,  389 
Noguchi  reaction  for,  271 
secondary  anemia  from,  276 
silver  impreganation  method  for, 

390 
spirochajta  pallida  in,  388 

refringens  in,  389 
treponema  pallidum  in,  ^i^^,  388, 
389 
stains  for,  390 
Wassermann   reaction   for,    264. 
See  also  Wassrrniann  reaction. 
Wright's  stain  for,  390 


T.KNiA,  347 

echinococcus,  346,  348,  349 
in  urine,  169 

elliptica,  351 

mediocanellata,  347 

saginata,  345,  346,  347 

solium,  345,  346,  346 
Tallquist's    estimation    of    hemo- 
globin, 191 

hemoglobinometer,  190,  191 
Tannin  in  urine,  138 
Tapeworm,  341,  345 

beef,  347 

dwarf,  350 

fish,  351 

in  feces,  313 

pork,  348 
Teichmann's  test  for  blood,  274 

for  hemoglobinuria,  125 
Telosporidia,  328,  338 
Temperature,  440 
Tertian  parasite,  249,  250,  256 
Test-breakfast,  Boas',  286 

Ewald's,  286 
Test-meals,  286 

Boas',  286 

Ewald's,  286 
Texas  fever,  Babesia  bigeminum  of, 

339 
Thoma-Zeiss  hemocytometer,  193, 
194 
cleaning  iru^trument,  199 
sources  of  error,  199 
technic,  195 
Thorn-apple  crystals  in  urine,  151 
Thread- worm,  355 


Thrush,  378 

Tick  fever,  Babesia  hominis  of,  339 

Tinea  versicolor,  384 

Tissue  bits  in  gastric  contents,  289 

Todd's  estimation  of  leukocytes  in 

leukemia,  211 
Toisson's    fluid    for    blood   count, 

198 
Tonsils,  diphtheria  of,  378 
Tdpfcr's  test  for  combined  hydro- 
chloric acid,  298 
for  free  hydrochloric  acid,  297 
for  total  acidity,  295 
Torfuge,  Wethcrill's,  139 
T.  O.  tuberculin,  428 
Toxic  absorption,  degree  of,  236 

leukocytosis,  207 
T.  R.  tuberculin,  428 
Trachea,    cylindric   cells   from,   in 

sputum,  61 
Trachoma,  383 
Transitional  leukocytes,  235 
Transitory  glycosuria,  108 
Transudates,  371 
Trematoda,  340,  341 
Trematodes,  340,  341 
Treponema,  ;i$^ 

pallidum,  333,  388,  389 
Giemsa's  stain  for,  390 
India-ink  method,  391 
silver    impregnation    method, 

390 
Wright's  stain  for,  390 
pertenue,  333 
Trichinella,  363 

spiralis,  324,  363,  364 
embryos  of,  in  blood,  257 
Trichiniasis,  diagnosis,  364 

parasite  of,  363 
Trichinosis,  eosinophilia  in,  239 
Trichloracetic  acid  test  for  albumin, 

102 
Trichocephalus,  364 

trichiurus,  364 
Trichomonas,  336 
intestinalis,  337 
pulmonalis,  337 
vaginalis,  336 
in  urine,  170 
Triple  phosphate  crystals  in  urine, 
148 
phosphates  in  urine,  87 


INDEX 


465 


Triple  stain,  Ehrlich's,  for  blood, 

221 
Tropical  dysentery,  entamoeba  his- 
tolytica in,  328 
Trypanosoma,  SSS 

brucei,  335 

cruzi,  334 

equiperdum,  335 

evansi,  335 

gambiensi,  334 
in  blood,  247 

lewisi,  334 
Trypanosomes,  333 

blepharoplast  of,  ^3$ 
Trypsin  in  feces,  Miiller's  test  for, 

322 
Tsuchiya's  method  for  albumin  in 

urine,  105 
Tube-casts  in  urine,  152 
Tubercle  bacillus.     See  also  Bacil- 
lus tuberculosis. 
Tuberculin,  428 

B.  E.,  428 

B.  F.,  428 

dosage  of,  428 

in  diagnosis,  429 

Calmette's     oph thai  mo- tuber- 
culin reaction,  430 
hypodermic  injection,  429 
Moro's  reaction,  430 
Von  Pirquet's  reaction,  430 

in  tuberculosis,  428 

reaction  of,  429 

T.  O.,  428 

T.  R.,  428 
Tuberculosis,    animal    inoculation 
in,  375 

Calmette's  reaction  in,  430 

diazo  reaction  in,  127 

Moro's  reaction  in,  430 

of  mouth,  380 

of  pharynx,  380 

pulmonary,  sputum  in,  66 

renal,  urine  in,  175 

secondary  anemia  from,  276 

tuberculin  in  diagnosis  of,  429 
in  treatment  of,  428 

vesical,  urine  in,  178 

Von  Pirquet's  reaction  in,  430 
Tubing  culture-media,  406 
Tumors,  malignant,  anemia  from, 

276 

30 


Tumors,  vesical,  urine  in,  178 
Tiirck's  ruling  for  blood  count  in 

leukemia,  209,  211 
Turpentine,  odor  of  urine  from,  73 
Two-slide  method  for  blood-films, 

217 
Typhoid  bacilllus,  417 
in  blood,  244 
technic,  245 
fever,  diazo  reaction  in,  127 
lymphocytes  in,  234 
secondary  anemia  from,  276 
vaccines  in,  428 
Widal  reaction  in,  127,  258 
Tyrosin  in  urine,  145,  146 


Uffelmann's  test  for  lactic  acid, 

292 
Ulcer,  gastric,  stomach  contents  in, 

306 
Ulcerations  of  mouth,  380 

of  pharynx,  380 
Uncinaria  duodenalis,  359 

life-history,  360 
Uncinariasis,  anemia  from,  276 

diagnosis  of,  361 

eosinophilia  in,  239 
Unstained  sputum,  40 
Urates,  amorphous,  in  urine,  72,  95, 

143 
in  mass,  160 
Urea  in  urine,  92 
decreased,  93 

estimation,  quantitative,  94 
increased,  92 
tests,  94 
Ureometer,  Doremus-Hinds',  94 
Uric  acid  crj'stals  in  urine,  142 
in  urine,  95 

Cook's  method,  96 
decreased,  96 

estimation,  quantitative,  96 
increased,  96 
Ruhemann's  method,  97 
Uricometer,  Ruhemann's,  96 
Urinary  albumin,  99 
crystals,  141,  142 
sediment,  examination,  138 
organized,  151 
transference  to  slide,  138 
imorganized,  141 


466 


INDEX 


Urinary    sediment,    unorganized, 
in  acid  urine,  141,  142 
in  alkaline  urine,  142,  148 
Urine,  68 

acctanilid  in,  132 

acetone    in,    118.  See    also 

Acelonuria. 
acid,  73 

unorganized  sediments  in,  141, 
.142 
acidity,  quantitative  estimation, 

74 
P'olin's  method,  74 

air  bubbles  in,  173 

albumin  in,  99.      See  also  Albu- 
minuria. 

alkaline,  unorganized  sediments 
in,  142,  148 

alkalinity  of,  73 
fixed,  74 
volatile,  74 

alkapton  bodies  in,  126 

ammonia  in,  97.      See  also  Am- 
monia in  urine. 

ammoniacal  decomposition,  74 

ammoniomagncsium     phosphate 
crystals  in,  148 

ammonium  urate  crystals  in,  151 

amorphous  phosphates  in,  149 

anguillula  aceti  in,  171 

animal  parasites  in,  169 

antipyrin  in,  132 

arsenic  in,  133 

Gutzeit's  test  for,  133 
Reinsch's  test  for,  133 

atropin  in,  133 

bacillus  tuberculosis  in,  168 
typhosus  in,  168 

bacteria  in,  72,  167 

bacterial  casts  in,  159 

Bence- Jones'  body  in,  108 
detection,  108 

bile  acids  in,  124 
Hay's  test,  124 
tests,  124 

bile  in,  123 

Gmelin's  test  for,  123 
Smith's  test  for,  123 

bile-pigment  in,  123 

bilifuscin  in,  123 

bilirubin  in,  123 

biliverdin  in,  123 


Urine,  blood  in,  72,  166 
blood-casts  in,  158 
blood-corpuscles  in,  165 
brick-dust  deposit  in,  72,  143 
bromids  in,  133 
bubbles  of  air  in,  173 
calcium  carbonate  in,  150 

oxalate  in,  144 

phosphate  crystals  in,  149 
casts  in,  152 

negative-staining,  154 
chemic  examination,  80 
chlorids  in,  82.    See  also  Chlorias 

in  urine. 
coffin-lid  crystals  in,  148 
color,  71 

composition  of,  68,  80 
constituents,  68 

abnormal,  99 

inorganic,  82 

normal,  80 

organic,  82 
cryoscopy  of,  79 
cylindroids  in,  160 
cystin  crystals  in,  146 
decreased,  71 

dextrose  in,  108.    See  also  Glyco- 
suria. 
diacetic  acid  in,  122.      See  also 

Diacetic  acid  in  urine. 
diazo  substances  in,  126 
dumb-bell  crystals  in,  150,  151 
earthy  phosphates  in,  149 
effect  of  drugs  on,  71,  132 
electric  conductivity,  79 
envelop  crystals  in,  144 
epithelial  casts  in,  158 

cells  in,  162,  163 
examination,  70 

chemic,  80 

microscopic,  138 

physical,  70 
extraneous  structures  in,  171 
fat-droplets  in,  172 
fat-globules  in,  147 
fatty  casts  in,  158 
fibers  in,  extraneous,  161,  172 

of  cotton,  161,  172 

of  linen,  161,  172 

of  silk,  161,  172 

of  wool,  161,  172 
fibrinous  casts  in,  157 


INDEX 


467 


Urine,  filariae  in,  170 
floaters  in,  169 
freezing-point,  79 
fruit-sugar  in,  117 
functional  tests  for,  78 
glucose  in,  108.     See  also  Glyco- 
suria. 
gonococci  in,  169 
gonorrheal  threads  in,  169 
granular  casts  in,  157,  158 
granule  cells  in,  compound,  162 
gravel  in,  142 
hairs  in,  161 
hip-roof  crystals  in,  148 
hyaline  casts  in,  154,  155 
hyphae  of  molds  in,  161 
in  calculus,  renal,  177 

vesical,  178 
in  chyluria,  148 
in  cystitis,  178 
in  diabetes  insipidus,  179 

mellitus,  179 
in  disease,  173 
in  hyperemia,  173,  174 
in  nephritis,  173,  175,  176 
in  pregnancy,  175 
in  pyelitis,  177 
in  renal  calculus,  177 

hyperemia,  173 

tuberculosis,  175 
in  vesical  calculus,  178 

tuberculosis,  178 

tumors,  178 
increased,  70 
indican  in,  90.     See  also  Indican 

in  urine. 
inorganic  constituents,  82 
iodin  in,  133 

irregular  epithelial  cells  in,  162 
lactose  in,  117 
lead  in,  134 

Lederer's  test,  134 
leucin  in,  145 
levulose  in,  117 
lycopodium  granules  in,  173 
-  melanin  in,  126 

tests  for,  126 
melanogen  in,  126 
mercury  in,  136 
methylene-lalue  test  for,  79 
micrococcus  ureae  in,  167 
microscopic  examination,  138 


Urine,  milk-sugar  in,  117 
mold  fungi  in,  172 
morphin  in,  136 
mucin  in,  106 
mucous  threads  in,  159 
normal  constituents,  &3 
nubecula  of,  72 
odor,  73 

organic  constituents,  82 
oxybutyric  acid  in,  123 
pavement  epithelial  cells  in,  163 
pentoses  in,  117 

Bial's  orcin  test,  118 
phenacetin  in,  132 
phenol  in,  136 
phenolphthalein  in,  137 
phloridzin  test  for,  79 
phosphates  in,  86,  148.    See  also 

Phosphates  in  urine. 
physical  examination,  70 
pigments  in,  71 

removal,  69 
polyhedral  cells  in,  162 
potassium   indoxyl   sulphate   in, 

90.    See  also  Indican  in  urine. 
proteins  in,  99 
proteoses  in,  106 

detection,  108 

primary,  106 

secondary,  106 
pseudocasts  in,  161 
purin  bodies  in,  95 

Cook's  method,  96 
pus  in,  72,  164 

Donne's  test,  72 
pus-casts  in,  159 
pus-corpuscles  in,  163 
quantity,  70 
quinin  in,  137 
reaction,  73 
red  blood-corpuscles  in,  165 

sand  in,  142 
resinous  drugs  in,  137 
retention  with  overflow,  70 
salicylates  in,  137 
salol  in,  137 
schistosomum  haetaatobium  in, 

170 
serum-albumin  in,  99 
serum-globulin  in,  99 
shadow  cells  in,  166 
sodium  urate  in,  144 


468 


INDEX 


Urine,  solids  in,  total,  76 
Hiiser's  method,  78 

specific  gravity,  74 

spermatozoa  in,  167 

squamous  epithelial  cells  in,  163 

starch-granules  in,  173 

sugars  in,  108 

suljihates  in,  88.      See  also  5m/- 
p/idtcs  in  urine. 

sul])huric  acid  in,  88 

sup])ression,  71 

ta.'nia  echinococcus  in,  169 

tannin  in,  138 

thorn-apple  crystals  in,  151 

total  solids  in,  76 

Hiiser's  method,  78 

transparency,  72 

trichomonas  vaginalis  in,  170 

triple  phosphate  crystals  in,  148 

tube-casts  in,  152 

tubercle  bacilli  in,  168 

tyrosin  in,  145,  146 

urates  in,  amorphous,  72,  95,  143 
in  mass,  160 

urea  in,  92.      See  also    Urea  in 
urine. 

uric-acid  crystals  in,  142 

uric  acid  in,  95.     See  also   Uric 
acid  in  urine. 

vinegar  eel  in,  171 

volatile  alkalinity  of,  74 

waxy  casts  in,  156 

yeast-cells  in,  171 
Urinometer,  Squibb's,  75 
Urinopyknomcter,  Saxe's,  76 


Vaccine  treatment,  264 
Vaccines,  419 

autogenous,  419 

bacterial,  Wright's,  264 

bottles,  420 

counting  of,  422 

dosage  of,  425 

clinical  method,  426 

in  infections,  426,  427 

in  malignant  endocarditis,  427 

in  typhoid  fever,  428 

injection  of,  425 
technic,  425 

method  of  use,  425 

preparation  of,  419 


Vaccines,  preparation  of,  diluting, 
424 
making  the  suspension,  421 
materials,  419 
obtaining  the  bacteria,  420 
sterilization,  422 
stock,  419 

therapeutic  indications,  426 
Vacuolated  leukocytes,  243 
Van  Gieson's  stain  for  Negri  bodies, 
Frothingham's  modification,  395 
Vegetable  cells  in  feces,  316 

fibers  in  feces,  316 
Vermidea,  340 

Vernal  catarrh,  eosinophilic  leuko- 
cytes in,  383 
Vesical  calculus,  urine  in,  178 
tuberculosis,  urine  in,  178 
tumors,  urine  in,  178 
Vincent's  angina,  379 
spirochaete  of,  331 
Vinegar  eel,  354 

in  urine,  171 
Vogel's  scale.    See  Frontispiece. 
Volume  index  of  blood,  200 

Larrabee's  method,  201 
method,  201 
Von  Fleischl's  estimation  of  hemo- 
globin, 185 
hemoglobinometer,  186 
Von  Pirquet's  reaction  in  tubercu- 
losis, 430 

Wassermann  reaction,  264 
bacteriolysis  in,  265 
effect  of  mercury  treatment  on, 

273 
of  salvarsan  treatment  on, 

273 
of  treatment  on,  273 
hemolysis  in,  265 
modifications,  271 
Noguchi's  modification,  271 
reagents  in,  269 
I  technic,  269 

value  of,  272 
Water-motor  centrifuge,  84 
Watery  blood,  181 
Waxy  casts  in  urine,  156 
Weights,  439 
I  Wetherill's  torfuge,  139 
I  Whip-worm,  364 


INDEX 


469 


White  blood-corpuscles,  180 
Widal  reaction,  258 

in  typhoid  fever,  127 

macroscopic,  261 

microscopic,  259 
Wires,  platinum,  397 
Wool  fibers  in  urine,  161,  172 
Working  distance  of  objective,  23 
Worms,  341,  345 

eosinophilia  as  symptom,  239 

pin-,  355 

round-,  353 

screw-,  366 

tape-,  345 

thread-,  355 

whip-,  364 
Wright  and  Kinnicutt's  estimation 

of  blood-plaques,  216 
Wright's  bacterial  vaccines,  264 

blood-stain,  222 
for  syphilis,  390 
in  cytodiagnosis,  372 


Wright's  capsule,  399 

method  of  obtaining  blood  in, 
258 
method  foi  measuring  opsonins, 

262 
stain  for  malarial  parasites,  254 

Xerosis  bacillus  in  eye,  382 

Yaws,  treponema  pertenue  of,  333 
Yeast-cells  in  gastric  contents,  302 

in  urine,  171 
Yeasts  in  sputum,  47 

Zappert  ruling  for  count  in  leu- 
kemia, 209 

Ziehl-Neelsen  method  for  bacillus 
tuberculosis  in  sputum,  51 

Zoomastigophora,  327,  330 

Zymogens,  284 


SAUNDERS'  BOOKS 


Practice,  Pharmacy, 
Materia  Medica,  Thera- 
peutics, Pharmacology, 
and  the  Allied  Sciences 

W.  B.  SAUNDERS  COMPANY 

92s  Walnut  Street  Philadelphia 

9,  Henrietta  Street  Covent  Garden,  London 

SAUNDERS'   SUCCESSFUL   PUBLISHING 

AS  is  well-known,  the  lists  of  most  publishers  contain  a 
number  of  books  that  have  never  paid,  and  for  which 
tlie  publisher  will  never  get  back  the  money  invested. 
Messrs.  \V.  B.  Saunders  Company  would  call  attention 
to  the  fact  that  they  have  no  such  works  on  their  list. 
In  all  the  years  of  their  business  experience  they  have 
never  published  a  book  at  a  loss.  This  they  confidently 
consider  a  most  remarkable  record,  and  submit  the  fact  to 
the  attention  of  the  profession  as  an  example  of  what  might 
justly  be  called  "  Successful  Publishing." 

A  Complete  Catalogue  of  our  Publications  will  be  Sent  upon  Request 


SAUXDEKS'    BOOK'S   O.V 


Musser    and    Kelly    on 
Treatment 


JUST  READY— IN  THREE  VOLUMES 
A  Handbook  of  Practical  Treatment.  By  77  eminent 
specialists.  Edited  by  John  H.  Musser,  M.  D.,  Professor  of 
Clinical  Medicine,  University  of  Pennsylvania ;  and  A.  O.  J. 
Kelly,  M.  D.,  Assistant  Professor  of  Medicine,  University  of 
Pennsylvania.  Three  octavo  voknnes  of  about  900  pages  each, 
illustrated.     Per  volume:     Cloth,    $6.00   net;    Half  Morocco, 

17-50  net. 

THE  EMINENT  CONTRIBUTORS 


Isaac  A.  Abt,  M.D. 
Sir  Clifford  AUbutt,  ^LD. 
James  M.  Anders,  M.U. 
John  F.  Anderson,  M.D. 
Le welly s  F.  Barker,  M.D. 
Joseph  C  Bloodgood,  M.D. 
George  Bliimer,  M.D. 
Sir  Lauder  Brunton,  NLD. 
Charles  W.  Burr,  M.D. 
Richard  C.  Cabot,  M.D. 
James  Carroll,  .M.D. 
JohnG.  Clark,  M.D. 
Rufus  L  Cole,  .M.D. 
Warren  Coleman,  M.D. 
Matthew  H.  Cryer,  .M.D. 
Clinton  T.  Dent,  M.D. 
Francis  X.  Dercum,  M.D. 
Geo.  E.  deSchweinitz,  M.D. 
George  Dock,  M.D. 
Isadore  Dyer,  M.D. 
David  L.  Edsall,  M.D. 
William  A.  Edwards,  M  D. 
.Arthur  W.  Elting,  M  D 
John  M.  T.  Finney,  M  D 
Charles  H.  Frazier,  M.D. 
M.   Howard  Fussell,  M.D. 


Thomas  B.  Futcher,  M.D. 
John  H.  Gibbon,  M.D. 
Joel  E.  Goldthwait,  M.D. 
Samuel  McC.  Hamill,  M.D. 
Hobart  A.  Hare,  M.D 
Charles  Harrington,  M.D. 
Ludvig  Hektoen,  M.D. 
Albion  Walter  Hewlett,  M.D 
Guy  Hinsdale,  M.D. 
Guy  L.  Hunner,  M.D. 
Chevalier  Jackson,  M.D. 
Henry  Jackson,  M.D. 
Theodore  C.  Janeway,  M.D. 
J.  H.  Jopson.  .M.D. 
A.  O.  J.  Kelly,  M.D. 
Maynard  I^dd.  M.D. 
Egbert  Lefevre,  M.D. 
James  Hendrie  Lloyd.  M.D. 
G.  Hudson  Makuen,  M.D. 
Charles  F.  Martin.  M.D. 
Edward  Martin,  M.D. 
Charles  H.  Mayo,  M.D. 
William  J    Mayo,  M.D. 
R.  Tait  McKenzie.  M.D. 
Herbert  C.  Moffit,  M.D. 
Jesse  .M.  Mosher,  M  D 


B.  G.  A.  Moynihan,  Esq. 
George  P.  Muller,  M.D. 
John  H.  Musser,  M.D. 
Edward  O.  Otis,  .\LD. 
Henry  K.  Pancoast,  M.D. 
Roswell  Park,  M.D. 
Richard  M.  Pearce,  M.D. 
Charles  W.  Richardson,  M.D. 
David  Riesman,  M.D. 
Milton  J.  Rosenau,  M.D. 
Joseph  Sailer,  ALD. 
J.  F.  Schamberg,  M.D. 
Henry  Sewell,  .M.D. 
Bertram  W.  Sippy,  M.D. 
William  G.  Spiller,  M  D. 
J.  Dufton  Steele.  ALD. 
Alfred  Stengel,  M.D. 
Charles  G.  Stockton,  .M.D. 
James  E.  Talley,  M.D. 
E.  W.  Taylor,  M.D. 
James  Tyson,  M.D. 
George  H.  Weaver,  M.D. 
J.  William  White,  M.D. 
AlfredC.  Wood,  M.D. 
Horatio  C.  Wood,  Jr.,  M.D. 


DIAGNOSIS  AND    TJiEATMENT 


Cabot's  Differential  Diag'nosis 

Differential  Diagnosis.  Presented  through  an  analysis  of 
383  Cases.  By  Richard  C.  Cabot,  M.D.,  Assistant  Professor  of 
Clinical  Medicine,  Harvard  Medical  School,  Boston.  Octavo  of 
753  pages,  illustrated.     Cloth,  ^5.50  net. 

THREE   PRINTINGS  IN  FOUR  MONTHS 

Dr.  Cabot's  work  takes  up  diagnosis  from  the  point  of  view  of  the  pre- 
senting symptom — tlie  symptom  in  any  disease  which  holds  the  foreground  in 
the  clinical  picture  :  the  principal  complaint.  It  groups  diseases  under  these 
symptoms,  and  points  the  way  to  proper  reasoning  in  coming  to  a  correct 
diagnosis.  It  works  backward  from  each  leading  symptom  to  the  actual 
organic  cause  of  the  symptom. 


Morrow's  Diagnostic  and 
Therapeutic   Technic 

Diagnostic  and  Therapeutic  Technic.  By  Albert  S. 
Morrow,  M.D.,  Adjunct  Professor  of  Surgery,  New  York  Poly- 
clinic. Octavo  of  775  pages,  with  815  original  line  drawings. 
Cloth,  $5.00  net. 

JUST  THE  WORK  FOR  PRACTITIONERS 

Dr.  Morrow's  new  work  is  decidedly  a  work  for  you — the  physician  en- 
gaged in  general  practice.  It  is  a  work  you  need  because  it  tells  you  just 
how  to  perform  those  procedures  required  of  you  every  day,  and  it  tells  you 
and  shows  you  by  clear,  «««/ line-drawings,  in  a  way  never  before  approached. 
.The  information  it  gives  is  such  as  you  need  to  know  every  day — transfusion 
and  infusion,  hypodermic  medication,  Bier's  hyperemia,  collection  and 
preservation  of  pathologic  material,  exploratory  punctures,  aspirations,  anes- 
thesia, etc.  Then  follow  descriptions  of  those  measures  employed  in  the 
diagnosis  and  treatment  of  diseases  of  special  regions  or  organs. 


SAUNDE/iS'    BOOK'S    OX 


Bonney  on    Tuberculosis 


Tuberculosis.  By  Sherman  G.  Bonney,  M.  D.,  Professor  of 
Medicine,  Denver  and  Gross  College  of  Medicine,  Denver.  Octavo 
of  955  pages,  with  243  original  illustrations.     Cloth,  $7.00  net. 

THE  NEW  (2d)  EDITION 

Dr.  Honney's  work  is  a  thorough  and  complete  treatise  of  the  entire  sub- 
ject of  tuberculosis.  The  section  on  Physical  Signs  of  Pulmonary  Tuber- 
culosis is  really  a  complete  monograph  on  the  physical  diagnosis  of  diseases 
of  the  chest.  Treatment  is  particularly  full  and  practical.  There  are  chapters 
on  prophylaxis  ;  open-air  treatment,  fully  illustrated  ;  diet  ;  sanitarium  and 
climatic  treatment  ;  therapeutic  measures  to  alleviate  distressing  symptoms; 
and  drug  and  vaccine  therapeutics. 

Maryland  Mediczd  Journal 

•■  Dr.  Bonney 's  book  is  one  of  the  best  and  most  exact  works  on  tuberculosis,  in  all  ils 
aspects,  that  has  yet  been  published." 

Anders  and  Boston's  Medical 
Diagnosis 

A  Text-Book  of  Medical  Diagnosis.  By  James  M.  An- 
ders, M.D.,  Ph.D.,  LL.  D.,  Professor  of  the  Theory  and  Prac- 
tice of  Medicine  and  of  Clinical  Medicine,  and  L.  Napoleon 
Boston,  M.D.,  Adjunct  Professor  of  Medicine,  Medico-Chirur- 
gical  College,  Philadelj)hia.  Octavo  of  11 75  pages,  with  443 
illustrations.     Cloth,  $6.00  net. 

JUST  READY 

This  new  work  is  tlesigned  expressly  for  the  general  practitioner.  The 
methods  given  are  practical  and  es^>ecially  adapted  for  quick  reference.  The 
diagnostic  methods  are  presented  in  a  forceful,  definite  way  by  men  who  have 
had  wide  experience  at  the  bedside  and  in  the  clinical  laboratory.  The  text 
IS  profusely  illustrated  with  original  pictures,  each  one  representing  some 
point  in  technic  or  some  diagnostic  sign.     It  is  a  work  for  every  practitioner. 


PRACTICE    OF  AfEDlCtNE 


Kemp    on 
Stomach    and    Intestines 


Diseases  of  the  Stomach  and  Intestines.  By  Robert 
Coleman  Kemp,  M.  D.,  Professor  of  Gastro-intestinal  Diseases 
at  the  New  York  School  of  Clinical  Medicine.  Octavo  of  766 
pages,  with  279  illustrations.     Cloth,  $6.00  net. 

FOUR  PRINTINGS  IN  FIFTEEN  MONTHS 

It  is  the  practitioner  who  first  meets  with  these  cases,  and  it  is  he  upon 
whom  the  burden  of  diagnosis  rests.  After  the  diagnosis  is  established,  the 
practitioner,  if  properly  equipped,  could  frequently  treat  the  case  himself 
instead  of  transferring  it  to  a  specialist.  This  work  is  intended  to  equip  the 
practitioner  with  this  end  in  view: 

The  Therapeutic  Gazette 

"  The  therapeutic  advice  which  is  g^ven  is  excellent.  Methods  of  physical  and 
chemical  examination  are  adequately  and  correctly  described." 


Deaderick  on   Malaria 


Practical  Study  of  Malaria.  By  William  H.  Deaderick, 
M.  D.,  Member  American  Society  of  Tropica)  Medicine. 
Octavo  of  402  pages,  illustrated.  Cloth,  $4.50  net;  Half 
Morocco,  ^6.00  net. 

A  WORK  LONG  NEEDED 

This  is  the  only  book  in  any  language  describing  the  third  cycle  of  the 
malarial  parasite — the  parthenogenetic  cycle.  The  chapters  on  diagnosis 
and  treatment  are  conspicuous  for  the  exactness  of  statement  and  the  intuitive 
way  in  which  the  author  has  grasped  the  needs  of  the  physician  and  supplied 
them. 

Frank  A.  Jones.  «M.  D., 

Professor  of  Clinical  Medicine  and  Physical  Diagnosis,  Memphis  Hospital  Medical 
College. 

"  Dr.  Deaderick's  book  is  up  to  date  and  the  subject  matter  is  well  arranged.  We 
have  been  waiting  for  many  years  for  such  a  work  written  by  a  man  who  sees  malaria  in 
all  its  forms  in  a  highly  malarious  climate." 


SAUNDERS'     BOOKS    ON 


Tousey's 

Medical  Electricity  anZ  X-Rays 

Medical  Electricity  and  the  X-Rays.  By  Sinclair 
TousEY,  M.  D.,  Consulting  Surgeon  to  St.  Bartholomew's 
Hospital,  New  York.  Octavo  of  1116  pages,  with  750  illustra- 
tions, 16  in  colors.     Cloth,  $7.00  net. 

ADOPTED  BY  THE  U.  S.  ARMY 

This  new  work  by  such  an  eminent  authority  is  destined  to  take  a  leading 
place  among  books  on  this  subject.  Written  primarily  for  the  general  prac- 
titioner, it  gives  him  just  the  information  he  wishes  to  have  regarding  the  use 
of  medical  electricity,  the  therapeutic  results  obtained,  etc.  At  the  same  time 
it  tells  the  specialist  how  the  most  eminent  electiotherai)eutists  are  securing 
results,  the  latest  authorities  in  every  country  having  been  consulted  for  de- 
tails of  practical  value.  The  work  gives  explicit  directions  for  the  care  and 
regulation  of  static  machines,  x-ray  tubes,  and  all  apparatus.  The  author 
tells  how  to  make  x-ray  pictures  by  a  practical  technic  easily  followed,  even 
though  the  operator  be  inexperienced  in  this  field.  Being  an 'authority  on 
dental  radiography,  the  chapters  on  this  side  of  the  subject  are  particularly 
imp>ortant  to  those  interested  in  dental  work. 
The  Therapeutic  Gazette 

"  Dr.  Tousey's  book  may  be  said  to  contain  practically  everything  in  regard  to  medical 
electricity,  and  by  '  everything  '  we  mean  not  only  a  discussion  of  elementary  facts,  largely 
physical  in  nature,  but  a  description  of  all  the  forms  which  can  be  employed." 

McKenzie   on  Exercise  in 
Education    and    Medicine 

Exercise  in  Education  and  Medicine.  By  R.  Tait 
McKenzie,  B.  A.,  M.  D.,  Professor  of  Physical  Education,  and 
Director  of  the  Department,  University  of  Pennsylvania.  Oc- 
tavo of  406  pages,  with  346  illustrations.     Cloth,  $3.50  net. 

ILLUSTRATED 

This  work  is  a  full  and  detailed  treatise  on  the  application  of  systematized 
exercise  in  the  development  of  the  normal  body  and  in  th#  correction  of  cer- 
tain diseased  conditions  in  which  gymnastics  have  proved  of  value. 

D.   A.    Sar^eantt    M.    D.,    Director  of  Hemenivay  Gymnasium,  Har^iard  University. 

"  It  cannot  fail  to  be  helpful  to  practitioners  in  medicine.  The  classification  of  athletic 
games  and  exercises  in  tabular  form  for  different  ages,  sexes,  and  occupations  is  the  work  of 
an  expert.     It  should  be  in  the  hands  of  every  physical  educator  and  medical  practitioner." 


THE    PRACTICE    OF   MEDICINE 


Anders* 
Practice    of    Medicine 

A  Text-Book  of  the  Practice  of  Medicine.  By  James 
M.  Anders,  M.  D.,  Ph.  D.,  LL.  D.,  Professor  of  the  Practice 
of  Medicine  and  of  Clinical  Medicine,  Medico-Chirurgical  Col- 
lege, Philadelphia.  Handsome  octavo,  1326  pages,  fully  illus- 
trated.    Cloth,  $5.50  net;  Half  Morocco,  $7.00  net. 

THE  NEW  (9th)  EDITION 

The  success  of  this  work  is  no  doubt  due  to  the  extensive  consideration 
given  to  Diagnosis  and  Treatment,  under  Differential  Diagnosis  the  jxiints  of 
distinction  of  simulating  diseases  being  presented  in  tabular  form.  In  this 
new  edition  Dr.  Anders  has  included  all  the  most  important  advances  in 
medicine,  keeping  the  book  within  bounds  by  a  judicious  elimination  of 
ol'solete  matter.      A  great  many  articles  have  also  been  rewritten. 

Wm.  E.  Quine,  M.  D.,   College  of  Physicians  and  Surgeons,  Chicago. 

"  I  consider  Anders'  Practice  one  of  the  best  single-volume  works  before  the  profession 
at  this  time,  and  one  of  the  best  text-books  for  medical  students." 


DaCosta's  Physical  Diagnosis 

Physical  Diagnosis.  By  John  C.  DaCosta,  Jr.,  Asso- 
ciate in  Clinical  Medicine,  Jefferson  Medical  College.  Octavo 
of  557  pages,  with  original  illustrations.     Cloth,  $3.50  net. 

ORIGINAL  ILLUSTRATIONS 

In  Dr.  DaCosta's  work  every  method  given  has  been  carefully  tested  and 
proved  of  value  by  the  author  himself.  Normal  physic.il  signs  are  explained 
in  detail  in  order  to  aid  the  diagnostician  in  determining  the  abnormal.  Both 
direct  and  differential  diagnoses  are  emphasized.  The  212  original  illustra- 
tions are  artistic  as  well  as  practical. 

Henry  L.  Eisner,   M.  D.,   Professor  of  Medicine,  Syracuse  University. 

"  I  have  reviewed  this  book  and  am  thoroughly  convinced  that  it  is  one  of  the  best 
ever  written  on  the  subject.     In  every  way  I  find'it  a  superior  production." 


SAUNDERS'    BOOKS   ON 


Sahli's  Diag(nostic  Methods 

Edited  by  Nath'l  Bowditch  Potter.   M.D. 


A  Treatise  on  Diagnostic  Methods  of  Examination. 

By  Prof.  Dr.  H.  Sahli,  of  Bern.  Edited,  with  additions,  by 
Nath'l  Bowditch  Potter,  M.D.,  Assistant  Professor  of  Clinical 
Medicine,  Columbia  University.  Octavo  of  1225  pages,  pro- 
fusely illustrated.     Cloth,  $6.50  net. 

THE  NEW  (2d)  EDITION,  RESET 
Lewellys  F.  Barker.  M.  D. 

Professor  of  Medicine,  Johns  Hopkins  UniTersHy 
"  1  am  delighted  with  it,  and  it  will  be  a  pleasure  to  recommend  it  to  our  students  in 
the  Johns  Hopkins  Medical  School." 

Friedenwald  and  Ruhrah 
on  Diet 


Diet  in  Health  and  Disease.  By  Julius  Friedenwald, 
M.  D.,  Professor  of  Diseases  of  the  Stomach,  and  John  Ruhrah, 
M.  D.,  Professor  of  Diseases  of  Children,  College  of  Physicians 
and  Surgeons,  Baltimore.  Octavo  of  764  pages.  Cloth, 
$4.00  net. 

NEW    (3d)    EDITION 

This  work  contains  a  complete  account  of  foodstuffs,  their  uses,  and 
chemical  composition.  Dietetic  management  in  all  diseases  in  which  diet 
plays  a  part  in  treatment  is  carefully  considered,  the  articles  on  diet  in  diseases 
of  the  digestive  organs  containing  numerous  diet-lists  and  explicit  instructions 
for  administration.  The  feeding  of  infants  and  children,  of  patients  before 
and  after  anesthesia  and  surgical  opention.s,  are  all  taken  up  in  detail. 

George  Dock.  M.  D., 

Professor  of  Theory  and  Practice  of  Medicine  and   Clinical  Medicine,  Tulane  Uni- 
versity  of  Louisiana. 

"  It  seems  to  me  that  you  have  prepared  the  most  valuable  work  of  the  kind  now  avail- 
able.    I  am  especially  glad  to  see  the  long  list  of  analyses  of  different  kinds  of  food  " 


PRACTICE    OF  MEDICINE. 


Oertel  on   Brig'ht's  Disease 

The  Anatomical  and  Histological  Processes  of  Bright 's 
Disease.  By  Horst  Oertel,  M.  D.,  Director  of  the  Riissell 
Sage  Institute  of  Pathology,  New  York.  Octavo  of  227  pages, 
with  44  text-illustrations  and  6  colored  plates.     Cloth,  $5.00  net. 

ILLUSTRATED 

These  lectures  deal  with  the  anatomic  and  histologic  processes  of  Bright' s 
disease,  and  in  a  somewhat  different  way  from  the  usual  manner.  Everywhere 
relations  are  emphasized  and  an  endeavor  made  to  reconstruct  the  whole  as  a 
unit  of  interwoven  processes.  In  the  preparation  of  his  lectures  the  author 
had  in  mind  a  twofold  aim  :  To  present  the  visual  picture  of  nephritis,  and 
to  prepare  the  proper  way  for  the  understanding  of  the  genesis  of  the  disease. 


Fenwick  on  Dyspepsia 

Dyspepsia.     By  William  Soltau  Fenwick,  M.  D.,  of  Lon- 
don.    Octavo  of  485  pages,  illustrated.     Cloth,  $3.00  net. 
Southern  Medical  Journal 

*'  The  suggestions  on  treatment  are  logical  and  practical,  being  particularly  helpful  in 
many  of  those  perplexing  types  so  often  encountered." 


Smith's  What  to  Eat  6  Why 

What  to  Eat  and  Why.  By  G.  Carroll  Smith,  M.D., 
Boston.     i2mo  of  312  pages.     Cloth,  $2.50  net. 

RECENTLY  ISSUED 

With  tliis  book  you  no  longer  need  send  your  patients  to  a  specialist  to 
be  dieted — you  will  be  able  to  prescribe  the  suitable  diet  yourself,  just  as  you 
do  other  forms  of  therapy.  Dr.  Smith  gives  "the  why"  of  each  statement 
he  makes.  It  is  this  knowing  wliy  which  gives  you  contidence  in  the  book, 
which  makes  you  feel  that  Dr.  Smith  knows. 


Slade's  Physical  Examination  O  Diagnostic  Anatomy 

Physical  Ex.'vmination  Axn  Dia(;N(«tic  Anatomy. — By  Charm. s 
B.  Sladk,  M.D.,  Chief  of  Clinic  in  General  Medicine,  University  and 
Bellevue  Hospital  Medical  College.  i2mo  of  146  pages,  illustrated. 
Cloth,  $1.25  net. 


lo  SAUNDERS'    BOOKS    ON 

AMERICAN  EDITION 

NOTHNAGEL'S    PRACTICE 

UNDER    THIi    EDITORIAL    SUI'EUVISION    OK 

ALFRED    STENGEL.    M.D. 

Professor  of  Medicine  in  the  University  of  Pennsylvania 


Typhoid  and  Typhus  Fevers 

\^y  I)K.  II.  CURSCHMANN,  of  Leipsic.  Edited,  with  additions,  by 
William  Oslkr,  M.  D.,  F.  R.  C.  P.,  Oxford  University,  Oxford, 
England.     Octavo  of  646  pages,  illustrated. 

Sm&llpox  (including  Vaccination) ,  Varicella,  Cholera 
Asiatica,  Cholera  Nostras,  Erysipelas,  Erysip- 
eloid, Pertussis,  and  Hay  Fever 

By  Dr.  H.  Immermann,  of  Uasle  ;  I)k.  Th.  von  JCkcensen,  of 
Tubingen  ;  Dr.  C.  Lieiskkmeistkr,  of  Tubingen  ;  Dr.  II.  Lenhartz, 
of  Hamburg;  and  Dr.  G.  Sticker,  of  Giessen.  The  entire  volume 
edited,  with  additions,  by  Sir  J.  \\.  Moore,  M.  D.,  F.  R.  C.  P.  I., 
Royal  College  of  Surgeons,  Ireland.     Octavo  of  682  pages,  illustrated. 

Diphtheria,  Measles,  Scarlet  Fever,  and  Rotheln 

By  William  P.  Northrup,  M.  D.,  of  New  York,  and  Dr.  Th. 
von  JOrgensen,  of  Tubingen.  The  entire  volume  edited,  with  additions, 
by  William  P.  Northrup,  M.  D.,  University  and  Bellevue  Hospital 
Medical  College.     Octavo  of  672  pages,  illustrated. 

Diseases  of  the  Bronchi,  Diseases  of  the  Pleura,  and 
Inflanmations  of  the  Lunges 

By  Dr.  F.  A.  Hoffmann,  of  Leipsic;  Dr.  O.  Rosenbach,  of 
Berlin;  and  Dr.  F.  Ai'FRECHT,  of  Magdeburg;  The  entire  volume 
edited,  with  additions,  by  John  H.  Musser,  M.  D.,  University  of  Penn- 
sylvania.    Octavo  of  1029  pages,  illustrated. 

Diseases  of  the  Pancreas,  Suprarenals,  and  Liver 

By  Dr.  T.  Oser,  of  Vienna;  Dr.  E  Nitsskr.  of  Vienna;  and  Drs. 
IT.  Quincke  and  G.  Hoppe-Sevi.er,  of  Kiel.  The  entire  volume 
edited,  with  additions,  by  Recinald  H.  Fit/.,  A.  M.,  M.  D.,  Harvard 
University;  and  Frederick  A.  Packard,  M.  D.,  Pennsylvania  and 
Children's  Hospitals,  Philadelphia.     Octavo  of  918  pages,  illustrated. 

PER  VOLUME:   CLOTH,  $5.00  NET;    HALF  MOROCCO.   $6.00  NET 


PRACTICE    OF  MEDICINE  ll 

AMERICAN   EDITION 

NOTHNAGEL'S    PRACTICE 


Diseases  of  the  Stomach 

By  Dr.  F.  Riegil,  of  Giessen.  Edited,  with  additions,  by  Charles 
G.  Stockton,  M.  D.,  University  of  Buffalo.     Octavo  of  835  pages. 

Second 

Diseases  of  the  Intestines  and  Peritoneum        Edition 

By  Dr.  Hermann  Nothnagel,  of  Vienna.  Edited,  with  additions, 
by  H.  D.  R0LI.ESTON,  M.  D.,  F.  R.  C.  P.,  St.  Georges  Hospital, 
London.     Octavo  of  1 100  pages,  illustrated. 

Tuberculosis  and  Acute  General  Miliary  Tuberculosis 

By  Dr.  G.  Cornet,  of  Berlin.  Edited,  with  additions,  by  Wai.ier 
B.  J.AMES,  M.  D.,  Columbia  University,  New  York.    Octavo  of  806  pages. 

Diseases  of  Blood  {Anemia,  Chlorosis,  Leukemia,  Pseudoleukemia) 

By  Dr.  P.  Ehrlich,  of  Frankfort-on-the-Main ;  Dr.  A.  Lazarus,  of 
Charloitenburg ;  Dr.  K.  von  Noorden,  of  Frankfort-on-the-Main;  and 
Dr.  Felix  Pinki;s,  of  Berlin.  The  entire  volume  edited,  with  addi- 
tions, by  Alfred  Stengel,  M.  D.,  University  of  Pennsylvania.  Octavo 
of  714  pages,  illustrated. 

Malaria,  Influenza,  and  Dengue 

By  Dr.  J.  Mannaberg,  of  Vienna,  and  Dr.  O.  Leichtenstern,  of 
Cologne.  The  entire  volume  edited,  with  additions,  by  Ro.nald  Ro.ss, 
F.  R.  C.  S.,  University  of  Liverpool  ;  J.  W.  \V.  Stephens,  M.  D., 
D.  P.  H.,  University  of  Liverpool  ;  and  Albert  S.  Grunbaum,  F. 
R.  C.  P.,  University  of  Liverjxjol.     Octavo  of  769  pages,  illustrated. 

Kidneys,  Spleen,  and  Hemorrhagic  Diatheses 

By  Dr.  H.  Senator,  of  Berlin,  and  Dr.  ^L  Litten,  of  Berlin.  The 
entire  volume  edited,  with  additions,  by  James  B.  Herkick,  M.  D., 
Rush  Medical  College.     Octavo  of  815  pages,  illustrated. 

Diseases  of  the  Heart 

By  Prof.  Dr.  Th.  von  JCrgensen,  of  Tubingen ;  Prof.  Dr.  L. 
Krehl,  of  Griefswald;  and  Prof.  Dr.  L.  von  Schrotter,  of 
Vienna.  The  entire  volume  edited,  with  additions,  by  George  Dock, 
M.  D.,  Tulane  University  of  Louisiana.      Octavo  of  848  pages. 

-       PER  VOLUME:  CLOTH.  $5.00  NET;  HALF  MOROCCO,  $6.00  NET 


Goepp's  State  Board  Questions 

State  Bo.-vrd  Questions  and  Answers.  By  R.  Max  Goepp, 
M.  D.,  Professor  of  Clinical  Medicine,  Philadelphia  Polyclinic.  Octavo 
of  715  pages.  Second  Edition.     Cloth,  ^(4.00  net. 

"  Nothing  has  been  printed  which  is  so  admirably  adapted  as  a  guide  and  self-quiz 
for  those  intending  to  take  State  Board  Examinations." — Pennsylvania  Medical 
Journal 


SAUNDERS'    BOOKS  ON 


Stevens*  Therapeutics  New  (5th)  Edition 

A  Text-Book  ok  Modern  Materia  Medica  and  Therapeutics. 
Hy  A.  A.  Stevens,  A.M.,  M.D.,  Lecturer  on  Physical  Diagnosis  in  the 
University  of  Pennsylvania.     Octavo  of  675  pages.     Cloth,  ^3.50  net. 

Dr.  Stevens'  Therapeutics  is  one  of  the  most  successful  works  on  the  subject  ever 
published.  In  this  new  edition  the  work  has  undergone  a  very  thorough  revision, 
and  now  represents  the  very  latest  advances. 

The  Medical  Record,  New  York 

'*  Among  the  numerous  treatises  on  this  most  important  branch  of  medical  practice, 
this  by  Dr.  Stevens  has  ranked  with  the  best." 

Butler's  Materia  Medico  New  (6th)  Edition 

A  Text-Book  of  Materia  Medica,  Therapeutics,  and  Pharma- 
cology. By  George  Y.  Buti.er,  Ph.G.,  M.I).,  Professor  and  Head 
of  the  Department  of  Therapeutics  and  Professor  of  Preventive  and 
Clinical  Medicine,  Chicago  College  of  Medicine  and  Surgery,  Medical 
Department  V^alpariso  University.  Octavo  of  702  pages,  illustrated. 
Cloth,  ;g4.oo  net ;  Half  .Morocco,  ;$5. 50  net. 

For  this  sixth  edition  Dr.  Butler  has  entirely  remodeled  his  work,  a  great  part  hav- 
ing been  rewritten.  All  obsolete  matter  has  been  eliminated,  and  special  attention 
has  been  given  to  the  toxicologic   and  therapeutic  effects  of  the  newer  compounds. 

Medical  Record,  New  York 

"  Nothing  has  been  omitted  by  the  author  which,  in  his  judgment,  would  add  to 
the  completeness  of  the  text." 

SoUmann's  Pharmacology  New  (2d)  Edition 

A  Text-Book  of  Pharmacology.  By  Torald  Sollmann,  M.D., 
Professor  of  Pharmacology  and  Materia  Medica,  Western  Reserve  Uni- 
versity.    Octavo  of  1070  pages,  illustrated.     Cloth,  $4.00  net. 

The  author  bases  the  study  of  therapeutics  on  systematic  knowledge  of  the  nature 
and  properties  of  drugs,  and  thus  brings  out  forcibly  the  intimate  relation  between 
pharmacology  and  practical  medicine. 

J.  F.  Fotherin^ham,  M.D.,  Trinity  Medical  College,  Toronto. 

"  The  work  certainly  occupies  ground  not  covered  in  so  concise,  useful,  and  scien- 
tific a  manner  by  any  other  text  1  have  read  on  the  subjects  embraced." 

Arny*s  Pharmacy 

Principles  of  Pharmacy.  By  Henry  V.  Arny,  Ph.  G.,  Ph.  D., 
Professor  of  Pharmacy  at  the  Cleveland  School  of  Pharmacy.  Octavo  of 
"75  P^g^s,  with  246  illustrations.      Cloth,  $5.00  net. 

George  Reimann,  Ph.  G.,  Secretary  of  the  Ne7v  York  state  Board  0/  Pharmacy. 

"  I  would  say  that  the  book  is  certainly  a  great  help  to  the  student,  and  I  think  it 
ought  to  be  in  thehands  of  every  person  who  is  contemplating  the  study  of  pharmacy." 


THERAPEUTICS  AND  MATERIA  MEDICA  13 


Hinsdale's   Hydrotherapy 

Hydrotherapy  :  A  Treatise  on  Hydrotherapy  in  General ; 
Its  Application  to  Special  Affections;  the  Technic  or  Processes 
Employed,  and  a  Brief  Chapter  on  the  Use  of  Waters  Internally. 
By  Guy  Hinsdale,  M.D.,  Fellow  of  the  Royal  Society  of  Great 
Britain.     Octavo  of  466  pages,  illustrated.     Cloth,  $3.50  net. 

The  treatment  of  disease  by  hydrotheiapeutic  measures  has  assumed  such 
an  important  place  in  medical  practice  that  a  good,  practical  work  on  the 
subject  is  an  essential  in  every  practitioner's  armamentarium.  This  new 
work  supplies  all  needs.  It  describes  fully  the  various  kinds  of  baths,  douches, 
sprays  ;  the  application  of  heat  and  cold ;  the  internal  use  of  mineral  waters 
and  all  other  procedures  included  under  hydrotherapeutic  measures.  Then 
the  use  of  hydrotherapy  in  the  various  diseases  is  detailed  concisely. 


Kelly's  Cyclopedia  of  American 
Medical  Biography 

Cyclopedia  of  American  Medical  Biography.  By  How- 
ard A.  Kelly,  M.  D.,  Professor  of  Gynecologic  Surgery  at  Johns 
Hopkins  University.  Two  octavos  of  750  pages  each,  with  por- 
traits. 

JUST  READY 

Dr.  Kelly,  in  these  two  handsome  volumes,  presents  concise,  yet  com- 
plete biographies  of  tliose  men  and  women  who  have  contributed  notewor- 
thily  to  the  advancement  of  medicine  in  America.  Dr.  Kelly's  reputation  for 
painstaking  care  assures  accuracy  of  statement.  There  are  about  one  thousand 
biographies  included.  

Swan's  Prescription-writing  and  Formulary 

Prescription  WRiriNG  and  Formul.ary.  By  John  M.  Swan, 
M.D.,  Director  Glen  Springs  .S*iitarium,  Watkins,  N.  Y.  l2mo  of  185 
pages.      Flexible  cloth,  $1.25  net. 

Stewart's    Pocket    Therapeutics    and    Dose- 
book  New  (4th)  Edition 
Pocket  Therapei:tics  and  Dose-book.     By  Morse  Siewart,  Jr., 
M.D.     32mo  of  263  pages.     Cloth,  ;$i.oo  net. 


14  SAUNDERS'    BOOKS   ON 

THE  BEST  /iinerican  standard 

Illustrated   Dictionary 

Just  Ready — The  New  (6th)  Edition,  Reset 

The  American  Illustrated  Medical  Dictionary.    By  W.  A. 

Nkw.man  Dokland,  M.  D.,  Editor  of  "The  American  Pocket 
Medical  Dictionary."  Octavo  of  975  pages.  Flexible  leather, 
54.50  net;  with  thumb  index,  $5.00  net. 

A  NEW  WORK— WITH  ADDED  FEATURES 

Howard   A.    Kellyt    M.  D.^  Johns  Hopkins  University,  Baltimore. 

"  Dr.  Borland's  dictionary  is  admirable.      It  is  so  well  gotten  up  and  of  such  conve- 
nient size.     No  errors  have  been  found  in  my  use  of  it." 


Thornton's  Dose-Book  Fourth  EdMon 

Dose-Book  and   Mamai.  of   PRKSCRiprioN-WRiTiNG.     By  E.  Q. 

Thornton,   M.  D.,  .Assistant   Professor  of   Materia    Medica,    Jefferson 

Medical    College,    Philadelphia.       Post-octavo,    392    pages,    illustrated. 

Flexible  leather,  S2.00  net. 

"  It  will  afford  me  much  pleasure  to  recommend  the  book  to  my  classes,  who  often 
fail  to  find  such  information  in  their  other  lexu-books." — C.  H.  Miller,  M.D., 
Profi-ssor  of  l^hatmacology.    Northwestern  University  Meiiical  School,  Chicago. 

Lusk    on    Nutrition  New  (2d)  Edition 

Elements  of  the  Science  of  Nutrition.  By  Graham  Lusk, 
Ph.D.,  Profes.-or  of  Physiology. in  Cornell  University  Medical  School. 
Octavo  of  402  pages.     Cloth,  J3.00  net. 

"I  shall  recommend  it  highly.  It  is  a  comfort  to  have  such  a  discussion  of  the 
subject."— Lewkllvs  K.  Hakkek,  M.  D.,  Pro/etsor  0/  the  PrincipUt  and  Practice 
0/ Meclicine,  Johns  Hopkins  University. 

Hatcher  and  Sollmann's  Materia  Medica 

A  1  E.KT-BooK  OF  M.VFERIA  Medica  :  including  Laboratory  Exer- 
cises in  the  Hi.stologic  and  Chemic  E.xamination  cf  Drugs.  By  Robert 
A.  n.ATCHER,  Ph.  (j.,  M.  D.  ;  and  ToRALD  Soi.lmann,  M.  D.  i2mo 
of  4 1 1  pages.     Flexible  leather,  $2  00  net. 

Bridi^e  on  Tuberculosis 

Tuber(  ui.osis.  By  Norman  Bridge,  A.  M.,  M.  D,  i2mo  of  302 
pages,  illustrated.     Cloth,  ^l  .50  net. 


MATERIA  MEDICA  AMD  THERAPEUTICS.  15 

American  Pocket  Dictionary  ^,^  JS;)^^Z 

The  American  Pocket  Medical  Dictionary.  Edited  by  W. 
A.  Newman  Borland,  M.D.  Flexible  leather,  with  gold  edges,  $1  00 
net ;  with  thumb  index,  $1.25  net. 

Eichhorst's  Practice  of  Medicine 

A  Text-Book  of  the  Practice  of  Medicine.  By  Dr.  H.  Eich- 
HORST,  University  of  Zurich.  Edited  by  A.  A.  Eshnkr,  M.  D.  Two 
octavos  of  6oo  pages  each,  illustrated.     Per  set :  Cloth,  ;$6.oo  net. 

Pusey  and  Caldwell  on  X-Rays  Second  Edition 

The  Practical  Application  of  the  Rontgen  Rays  in  Thera- 
peutics and  Diagnosis.  By  William  Allen  Pusey,  A.  M.,  M.  D., 
and  Eugene  W.  Caldwell,  B.  S.  Octavo  of  625  pages,  with  200 
illustrations.     Cloth,  $5.00  net. 

Cohen   and   Eshner's    Diagnosis.     Second  Revised  Edition 

Essentials  of  Diagnosis.  By  S.  Solis-Cohen,  M.  D.,  and  A.  A. 
EsHNER,  M.  D.  Post-octavo,  382  pages  ;  55  illustrations.  Cloth,  $1.00 
net.      In  Saunders^  Question- Compend  Series. 

Seventh 

Morris'  Materia  Medica  and  Therapeutics         Edition 

Essentials  of  Materia  Medica,  Therapeutics,  and  Prescrip- 
tion-Writing. By  Henry  Morris,  M.  D.  Revised  by  W.  A.  Bas- 
TEDo,  M.  1).,  Instructor  in  Materia  Medica  and  Pharmacology,  Columbia 
University.     1 2mo,  303  pages.     Cloth,  ;^i.oo  net.    Saunders'' Conipends. 

iVilliams'  Practice  of  Medicine 

Essentials  of  the  Practice  of  Medicine.  By  W.  R.  Williams, 
M.D.,  formerly  Lecturer  on  Hygiene  and  Instructor  in  Medicine,  Cornell 
University,  N.  Y.  i2mo  of  460  pages.  Double  number,  ^1.75  net.  In 
Saunders'  Question-  Compend  Series. 

Sarton  and  Wells*  Thesaurus 

A  Thesaurus  of  Medical  Words  and  Phrasics.  By  Wilfred  M. 
Barton,  M.  D.,  and  Walter  A.  Wells,  M.  D.  i2mo  of  534  pages. 
Flexible  leather,  $2.50  net ;  with  thumb  index,  gj.oo  net. 

Mathews'  How  to  Succeed  in  Practice 

How  TO  Succeed  in  the  Practice  of  Medicine.  By  Joseph  M. 
Mathews,  M.  D.,  LL.D.,  President  American  Medical  Association, 
1898-99.      i2mo  of  215  pages,  illustrated.      Cloth,  $1.50  net. 

Boston's  Clinical  Diagnosis  Second  Edition 

Clinical  Diagnosis.  By  Laboratory  Methods.  By  L.  Napoleon 
BosioN,  A.  M.,  M.  D.,  Adjunct  Professor  of  Medicine,  Medico- 
Chirurgical  College,  Philadelphia.  Octavo  of  563  pages,  with  330  illus- 
trations, many  in  colors.     Cloth,  54-00  net. 

Arnold's  Medical  Diet  Charts 

Medical  Diet  Charts.  Prepared  by  H.  D.  Arnold,  M.  D., 
Professor  of  Clinical  Medicine,  Tufts  Medical  College,  Bo.ston.  Single 
charts,  5  cents;  50  charts,  $2.00  net;  500  charts,  $\^.QO  net;  lOOO 
charts,  $30.00  net. 


i6  SAUNDERS'    BOOKS    ON   PRACTICE,   Etc. 


Saunders'   Pocket  Formulary  New  (9th)  Edition 

Sauni:)ERs'  Pocket  Medical  Formulary.  IJy  William  M. 
I'oWELL,  M.  D.  Containing  1900  formulas  from  the  best-known 
authorities.  In  flexible  leather,  with  side  index,  wallet,  and  flap. 
$1.75  net. 

Jakob  and  Eshner's  Internal  Medicine  and  Diagnosis 

Atlas  and  Epitome  ok  Internal  Medicine  and  Clinical  Diag- 
nosis. By  Dr.  Chr.  Jakob,  of  Erlangen.  Edited,  with  additions,  by 
A.  A.  EsHNER,  M.  D.  182  colored  figures  on  68  plates,  64  text-cuts, 
259  pages  of  text.     Cloth,  ^jSj-OO  net.     In  Saiindeii  Hand-Atlas  Series. 

Lockwood's  Practice  of  Medicine    Revisef^nd'ESll^S 

A  Manual  ok  the  Practice  ok  Medicine.  By  Geo.  Roe  Lock- 
wood,  M.  D.,  Attending  Physician  to  tlie  Bellevue  Hospital,  New  York 
City.     Octavo,  847  pages,  illustrated.     Cloth,  $\.(X)  net. 

Gould  and   Pyle's  Curiosities  of  Medicine 

Anomalies  and  Curiosities  of  Medicine.  By  George  M.  Gould. 
M.  I).,  and  Walter  L.  Pyle,  M.  D.  Octavo  of  968  pages,  295  en 
gravings,  and  12  full-page  plates.     Cloth,  $3.00  net. 

Jelliffe's   Pharmacognosy 

An  Introduction  to  Pharmacognosy.  By  Smith  Ely  Jelliffe, 
Ph.  D.,  M.  D.,  Columbia  University,  New  York.  Octavo  of  265  pages, 
illustrated.     Cloth,  ;5S2.so  net. 

Stevens*   Practice   of  Medicine  New  (8th)  Edition 

A  Manual  ok  the  Practice  of  Medicine.  By  A.  A.  Stevens, 
A.  M.,  M.  D.,  Professor  of  Therapeutics  and  Clinical  Medicine,  Woman's 
Medical  College,  Philadelphia.  i2mo,  558  pages,  illustrated.  Elexil)le 
leather,  $2.^0  net. 

Camac's  Epoch-Making  Contributions 

Epoch-making  Contributions  to  Medicine  and  Surgery.  By 
C.  N.  B.  Camac,  M.  1).,  of  New  York  City.  Octavo  of  450  pages, 
with  pi  rlraits.     Artistically  lx>und,  ;^4.CX>  net. 

Todd's  Clinical  Diagnosis 

Manual  of  Clinical  Diagnosis.  By  James  Campbell  Todd,  M. 
D.,  Associate  Professor  of  Pathology,  Denver  and  Gross  College  of 
Medicine,  Denver.  i2mo  of  319  pages,  illustrated.  Flexible  leather, 
$2.00  net. 

RoUeston  on  the  Liver 

Diseases  of  the  Liver,  Gall-bladder,  and  Bile-ducts.  By 
H.  D.  Rolleston,  M.  D.  (Cantab.),  London.  Octavo  of  794  pages, 
illustrated,  including  a  number  in  colors.     Cloth,  $6.00  net. 


University  of  California 

SOUTHERN  REGIONAL  LIBRARY  FACILITY 

405  Hilgard  Avenue,  Ilos  Angeles,  CA  90024-1388 

Return  this  material  to  the  library 

from  which  it  was  borrowed. 


<f/ 


A     000  499  536     i 


. ^ 

T63hc 

1912 

Todd. 

Clinical  diagnosis 

CALIFORNIA  COLLEGE  OF  MEDICINE  LIBRARY 

UNIVERSITY  OF  CALIFORNIA,  IRVINE 

IRVINE,  CALIFORNIA  92664 


PIIIWTKD  IM  v.*.*-